Disease treatment via antimicrobial peptides or their inhibitors

ABSTRACT

Provided are methods for the treatment of disease and promotion of healing that include providing a therapeutically effective amount of a mammalian antimicrobial peptide (AMP) or analog thereof, in particular a cathelicidin or cathelicidin fragment or cathelicidin analog, thereby treating the disease in the subject in need thereof. Also provided are specific analogs or fragments of cathelicidin that function as agonists, as do endogenous cathelicidins, or as either dominant negatives or as inhibitors to endogenous cathelicidin or to other endogenous AMPs or that compete with pro-inflammatory agents or fragments of AMPs on cognate receptors without inducing disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of prior application Ser. No.13/459,191 filed on Apr. 29, 2012, which in turn is a divisional of Ser.No. 12/173,344, filed Jul. 15, 2008 (now U.S. Pat. No. 8,202,835 issuedon Jun. 19, 2012), which is in turn, a continuation-in-part of U.S.application Ser. No. 10/539,558 filed Jun. 17, 2005 (U.S. Pub. No.2006/0115480 A1) and also claims priority to each of Israel applicationserial nos. 184611 filed Jul. 15, 2007 and 187627 filed Nov. 26, 2007,the disclosures of which are incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

The invention relates to the field of mammalian antimicrobial peptides(AMPs) and their use in the treatment of disease.

The Sequence Listing submitted in text format (.txt) on Jul. 18, 2012,named “SEQ.txt”, (created on Tuesday, Mar. 21, 2013, 36.2 KB), isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to methods of treating diseases usinganti-antimicrobial peptide (AMP) and/or AMP-like molecule (AML) and inparticular cathelicidin type AMPs, and to methods of identifyingcompounds capable of regulating, decreasing or increasingactivities/levels of AMPs/AMLs so as to enable treatment of diseases.More particularly, the present invention relates to methods of treatingdiseases by using cathelicidin or cathelicidin fragments or cathelicidinanalogs or compounds capable of regulating the levels/activity ofcathelicidin, such diseases including dysregulated cellproliferation/differentiation leading to bone loss or degradation,osteoporosis osteoarthritis, or to other autoimmune diseases such asmultiple sclerosis, arthritis, psoriasis, and to malignancies such ascarcinomas, which are associated with inflammation, to metabolicdiseases, obesity, insulin resistance, diabetes type 2, diabetes type 1and related diseases. Also, the present invention relates to methods ofidentifying compounds capable of regulating levels of cathelicidin orother AMPs or to increasing or to decreasing activity/levels of AMPs soas to enable treatment of diseases including autoimmune and inflammatorydiseases such as, multiple sclerosis, arthritis, metabolic disorderssuch as diabetes, obesity and malignant diseases such as carcinomas,which are associated with inflammation, dysregulated cellproliferation/differentiation, angiogenesis and/or metastasis.

Both inhibiting endogenous cathelicidin based peptides or other AMPs aswell as the use of such cathelicidin based peptides or analogs ofcathelicidin peptides are effective modes of treatments for disease. Aswas demonstrated in WO 2004-056307 filed by the present inventors andincorporated herein, cathelicidins are immune regulators and are overexpressed locally in autoimmune diseases. They are also expressedsystemically through bone marrow such that normal plasma concentrationsaverage around 1.2 ug/ml to 1.5 ug/ml (Journal of Immunological Methods206_(—)1997.53-59). Regulation of their expression is essential forhomeostasis. AMPs are involved is skewing dendritic cell activationbetween Th1 and Th2 inflammatory processes via Toll-like receptors andtherefore are involved in homeostasis (J Immunol. 2004 Jan. 15;172(2):1146-56). Controlling or maintaining such homeostasis isperformed by either increasing or decreasing of level/activity betweenthe various AMPs.

Cathelicidins are mainly expressed by Vitamin D3 (calcitriol), viavitamin D3 receptor elements (VDRE) and Vitamin D3 itself has amodulating influence on cathelicidin expression both as an agonist viacalcitriol/VDRE and by a negative feedback mechanisms (Marshall TBioEssays 30:173-182, 2008). This VDRE/cathelicidin pathway is unique tohumans and furry/haired animals such as rodents for example whose skinis less exposed to sunlight do not possess this pathway. As shown indata included in this invention for the first time relative to priorart, cathelicidin forms a major immune regulator for diseases which areknown to be also regulated by vitamin D3. These include amongst others,bone loss in Periodontitis (which is associated with low vitamin D andlow cathelicidin), Obesity, Type 2 Diabetes mellitus type 1 and type 2(which is associated with low vitamin D and Toll like receptor 4, whichcathelicidin inhibits), Atherosclerosis (low vitamin D association),Hypertension (low vitamin D association), Asthma and Allergy (lowvitamin D association), Osteoporosis and Ostepenia (low vitamin Dassociation), Multiple Sclerosis (low vitamin D association), Rheumatoidarthritis (low vitamin D association), Autoimmune Diseases such asCrohn, Type 1 Diabetes (low vitamin D association), Schizophrenia (lowvitamin D association), Muscle wasting disease including age associatedmuscle wasting (low vitamin D association as well as beta defensinoverexpression), Cancer (low vitamin D association as well asCathelicidin and beta defensin overexpression), Depression (low vitaminD association), Skin inflammation including Psoriasis (treated withvitamin D analogues), Tubeculosis and Influenza (low vitamin Dassociation), Chronic Pain (low vitamin D association), Osteoartheritis(low vitamin D association), The Common Cold and other known diseases(The Breast Journal, Volume 14 Number 3, 2008 255-260, PhotochemPhotobiol. 2008 March-April; 84(2):356-65) associated with vitamin D3,commonly known as the “Sunshine vitamin” and inappropriately called avitamin but is in fact a hormone. Data as presented in this inventionindicate a common pathway of disease regulation between cathelicidin andvitamin D3. For this reason, the inventor reasons that diseases such asschizophrenia and depression which cannot be modeled suitably by animalsare also regulated by cathelicidin.

Diseases, such as malignant, autoimmune, and allergic diseases, whichare associated with biological processes such as inflammation,dysregulated cell proliferation/differentiation, and dysregulated cellproliferation/differentiation balance include a vast range of highlydebilitating and/or lethal pathologies of great economic impact, forwhich no satisfactory treatment methods are presently available. Forexample autoimmune diseases represent diseases of major clinical andeconomic impact. These include major diseases such as psoriasis,rheumatoid arthritis, type I diabetes, inflammatory bowel diseases, andmultiple sclerosis for which no satisfactory treatment methods areavailable. Similarly, malignant diseases, such as skin carcinoma, breastcarcinoma, colon carcinoma, head and neck carcinoma, hepatic carcinoma,lung carcinoma, renal cell carcinoma, urinary bladder carcinoma, and thelike, represent numerous lethal diseases for which no satisfactorytreatment methods are available.

There is an urgent and long-felt need for optimal methods of treatingsuch diseases which are associated with inflammation, dysregulatedcell/tissue proliferation/differentiation and autoimmunity.

The epithelial lining of the skin, gastrointestinal tract and bronchialtree produces a number of peptides with antimicrobial activities termedantimicrobial peptides (AMPs), which appear to be involved in bothinnate host defense and adaptive immune responses (Yang D. et al., 2001.Cell Mol Life Sci. 58:978-89). AMPs are cationic peptides which displayantimicrobial activity at physiological concentrations under conditionsprevailing in the tissues of origin. AMP synthesis and release isregulated by microbial signals, developmental and differentiationsignals, cytokines and in some cases neuroendocrine signals in atissue-specific manner. Their mode of action is unknown, however theleading theory claims that permeabilization of target membranes is thecrucial step in AMP-mediated antimicrobial activity and cytotoxicity.AMPs are classified into two major groups in humans; cathelicidins anddefensins. AMPs appear to have common characteristics that enable themto affect mammalian cells in a way that does not necessarily functionthrough a ligand-receptor pathway, and that, being small, and highlyionic or hydrophobic or structurally amphiphilic, AMPs can bindmammalian cell membranes. They are able to penetrate through the cellmembrane to the cytoplasm. For example, it was shown that granulysinpenetrates and damages human cell membranes dependent upon negativecharge (J. Immunol., 2001, 167:350-356). At high concentrations they arecytotoxic to cells, they tear through the membrane causing lysis orapoptosis. Likewise they are able to change the charge density of theinner membrane by the very fact that they have charge, are small and aredistributed around the cell membrane from the outer surface of themembrane.

Cathelicidins contain a conserved “cathelin” precursor domain. Theirorganization includes an N-terminal signal peptide, a highly conservedprosequence, and a structurally variable cationic peptide at theC-terminus. The prosequence resembles cathelin, a protein originallyisolated from porcine neutrophils as an inhibitor of cathepsin L (hence,the name cathelin). The 37 amino acid-long human cathelicidin,LL-37/hCAP18 has a hydrophobic N-terminal domain in an α-helicalconformation, particularly in the presence of negatively charged lipids.In a step essential for its activation, LL-37 is enzymatically cleavedfrom the C-terminus of hCAP18 precursor via enzymes such as neutrophilelastase and proteinase 3. LL-37 functions in synergy with other AMPs,and can directly activate host cells. Inappropriate cleavage of thecathelicidin hCAP18 pro-peptide by endogenous proteases can producepro-inflammatory fragments of the cathelicidin (Nat Med. 2007 August;13(8):975-80). At the same time, correct cleavage via appropriateendogenous protease processing will produce the anti-inflammatorycathelicidin analogs and peptides. Thus, a method for regulatinginadequate processing of cathelicidin is required as well as a method ofusing the anti-inflammatory analogs or fragments to the cathelicidinpeptides or pro-peptide is described and exemplified below.

The ability of cathelicidins such as LL-37 to both kill bacteria andregulate immune responses is a characteristic of numerous AMPs. Thepeptide can influence host immune responses via a variety of cellularinteractions, for example, it has been suggest to possibly function as achemoattractant by binding to formyl-peptide-receptor-like-1 (FPRL-1).LL-37 can recruit mast cells, and then be produced by the mast cell tokill bacteria.

AMPs exert their effects either individually or as the resultant effectof multiple AMPs. For example, in the menstrual cycle there is a monthlycycle-dependent expression of various AMPs (King A. E. et al., 2003. J.Reprod. Immunol. 59:1-16). For example, there is higher expressionduring the menstrual cycle of beta-defensin-2 in the menstrual stage,beta-defensin-4 in the proliferative stage, beta-defensin-3 in the earlysecretory stage, beta-defensin-1 in the mid secretory stage, andbeta-defensin-3 in the late secretory stage. It has been suggested thatmaintaining the balance between the AMPs is essential for normalproliferation, differentiation and in the specific example of menstrualcycle for development. In light of the apparent roles of AMPs and mostimportantly of cathelicidin as was demonstrated in this and the formerpatent application (number WO 2004-056307) of the current inventor,cathelicidin is associated with inflammation, dysregulated cellproliferation/differentiation, dysregulated cellproliferation/differentiation balance, angiogenesis metastasis, and/orepithelial wounds, the inventor hypothesized that an optimal strategyfor treating such diseases would be via methods involving decreasing thelevels/activity of such AMPs/AMLs, and/or via methods involvingadministering such AMPs/AMLs or enhancing their expression.

The prior art approaches relating to such methods involve the previousapplication of the inventors in WO 2004-056307 which show thatcathelicidin is an immune regulator in-vivo and therefore poses a targetin treating autoimmune diseases.

The current application provides in-vivo data for specific diseases suchas metabolic diseases and low grade inflammatory diseases, obesity,insulin resistance, diabetes type 2, type 1 diabetes, insulin relateddiabetes, osteoporosis, periodontitis, osteoarthritis, arthriticdiseases, rheumatologic diseases such as rheumatoid arthritis,ankylosing spondylitis, gout and systemic lupus erythematosus, as wellas multiple sclerosis, neurological and central nervous system diseasesas well as osteoporosis.

In particular, the current invention shows in-vivo the use ofcathelicidin or cathelicidin analogs in the treatment of said diseases.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of treating a medical condition, such as a disease, in a subjectin need of treatment thereof, the method comprising providing to thesubject a therapeutically effective amount of a compound in particular acathelicidin peptide or fragment analog thereof, being capable oftreating the disease in the subject in need thereof or of regulating, orincreasing or decreasing an activity and/or level of an antimicrobialpeptide (AMP) and/or AMP-like molecule, thereby treating the disease inthe subject in need thereof.

According to further features in preferred embodiments of the inventiondescribed below, administering the compound to the subject is effectedby exposing a location of the subject to a carrier which includes thecompound at a concentration selected from a range of about 50 nanogramsper milliliter to about 2 milligram per milliliter.

According to still further features in the described preferredembodiments, administering the compound to the subject is effected byadministering to the subject a plurality of doses of the compoundselected from a range of 2 doses to 30 doses, wherein each inter doseinterval of the plurality of doses is selected from a range of about 2.4hours to about 30 days.

According to still further features in the described preferredembodiments, administering the compound to the subject is effected via aroute selected from the group consisting of the topical, intravenous,intranasal, transdermal, intradermal, oral, buccal, parenteral, rectaland inhalation route.

According to still further features in the described preferredembodiments, the disease is associated with a biological process in acell and/or tissue, wherein the biological process is selected from thegroup consisting of growth, differentiation, autoimmunity orinflammation.

According to still further features in the described preferredembodiments, the subject is human.

According to another aspect of the present invention there is providedan article of manufacture comprising packaging material and apharmaceutical composition, the article of manufacture being identifiedfor treatment of a disease being associated with a biological process ina cell and/or tissue, the biological process being selected from thegroup consisting of growth, differentiation or diseases associated withinflammation or autoimmunity; the pharmaceutical composition comprisinga pharmaceutically acceptable carrier and, as an active ingredient, acompound being capable of regulating an activity and/or level of anantimicrobial peptide (AMP) and/or AMP-like molecule.

According to further features in preferred embodiments of the inventiondescribed below, the pharmaceutically acceptable carrier is selected soas to enable administration of the pharmaceutical composition via aroute selected from the group consisting of the topical, intranasal,transdermal, intradermal, intravenous, oral, buccal, parenteral, rectaland inhalation route.

According to still further features in the described preferredembodiments, the pharmaceutical composition is formulated as a solution,suspension, emulsion or gel.

According to still further features in the described preferredembodiments, the pharmaceutical composition is composed so as to enableexposure of a cell and/or tissue of a subject having the disease to thecompound at a concentration selected from a range of about 50 nanogramsper milliliter to about 1 milligram per milliliter.

According to still further features in the described preferredembodiments, the pharmaceutical composition is further identified foradministration to a subject of a plurality of doses of thepharmaceutical composition selected from a range of 2 doses to 30 doses,wherein each inter dose interval of the plurality of doses is selectedfrom a range of about 2.4 hours to about 30 days

According to still further features in the described preferredembodiments, the cell and/or tissue is selected from the groupconsisting of skin cells, bone cells beta cells and synovial tissue.

According to still further features in the described preferredembodiments, the disease is selected from the group consisting of anautoimmune disease, a bone resorption disease, a neurological disease, ametabolic disease including diabetes, obesity, and a diabetes relateddisease.

According to yet another aspect of the present invention there isprovided a method of regulating a biological process in a cell and/ortissue, the method comprising exposing the cell and/or tissue to acompound in particular a cathelicidin peptide or its analog, beingcapable of regulating the biological process in the cell and/or tissueor of increasing or decreasing an activity and/or level of anantimicrobial peptide (AMP) and/or AMP-like molecule, thereby regulatingthe biological process in the cell and/or tissue.

According to further features in preferred embodiments of the inventiondescribed below, exposing the cell and/or tissue to the compound (suchas for example, a cathelicidin peptide or its analog) effected byproviding said compound to a subject.

According to still further features in the described preferredembodiments, the providing to the subject the compound is effected byadministering the compound to the subject and/or by expressing thecompound in the subject.

According to still further features in the described preferredembodiments, the exposing the cell and/or tissue to the compound iseffected by exposing the cell and/or tissue to the compound at aconcentration selected from a range of about 50 nanograms per milliliterto about one milligram per milliliter.

According to still further features in the described preferredembodiments, the cell and/or tissue is bone or nerve tissue or synovialtissue, wherein the exposing the cell and/or tissue to the compound(such as for example, a cathelicidin peptide or its analog) is effectedby exposing the cell and/or tissue to the compound at a concentrationselected from a range of about 0.4 microgram per milliliter to about 100micrograms per milliliter.

According to still another aspect of the present invention there isprovided a method of identifying a compound being capable of regulatinga biological process in a cell and/or tissue, the method comprising: (a)exposing the cell and/or tissue to a test compound which is: (i) capableof decreasing an activity and/or level of an antimicrobial peptide (AMP)and/or AMP-like molecule, and/or (ii) the AMP and/or AMP-like molecule;and (b) evaluating a capacity of the test compound to regulate thebiological process in the cell and/or tissue, thereby identifying thecompound being capable of regulating the biological process in the celland/or tissue.

According to still further features in the described preferredembodiments, the cell and/or tissue is a cultured cell and/or tissue.

According to still further features in the described preferredembodiments, the cell and/or tissue is derived from a human.

According to still further features in the described preferredembodiments, the exposing the cell and/or tissue to the test compound iseffected by providing the test compound to a subject.

According to still further features in the described preferredembodiments, the exposing the cell and/or tissue to the test compound iseffected by exposing the cell and/or tissue to a cell which produces thetest compound.

According to still further features in the described preferredembodiments, the cell which produces the test compound is a B-cellhybridoma.

According to still further features in the described preferredembodiments, the providing the test compound to the subject is effectedby administering the test compound to the subject and/or by expressingthe test compound in the subject.

According to still further features in the described preferredembodiments, administering the test compound to the subject is effectedvia a route selected from the group consisting of the topical,intranasal, intravenous, transdermal, intradermal, oral, buccal,parenteral, rectal and inhalation route.

According to still further features in the described preferredembodiments, the test compound is selected from the group consisting of:(a) a molecule capable of binding the AMP and/or AMP-like molecule; (b)an enzyme capable of cleaving the AMP and/or AMP-like molecule; (c) ansiRNA molecule capable of inducing degradation of an mRNA encoding theAMP and/or AMP-like molecule; (d) a DNAzyme capable of cleaving an mRNAor DNA encoding the AMP and/or AMP-like molecule; (e) an antisensepolynucleotide capable of hybridizing with an mRNA encoding the AMPand/or AMP-like molecule; (f) a ribozyme capable of cleaving an mRNAencoding the AMP and/or AMP-like molecule; (g) a non-functional analogof at least a functional portion of the AMP and/or AMP-like molecule;(h) a molecule capable of inhibiting activation or ligand binding of theAMP and/or AMP-like molecule; and (i) a triplex-forming oligonucleotidecapable of hybridizing with a DNA encoding the AMP and/or AMP-likemolecule.

According to still further features in the described preferredembodiments, the molecule capable of binding the AMP and/or AMP-likemolecule is an antibody or an antibody fragment.

According to still further features in the described preferredembodiments, the antibody fragment is selected from the group consistingof a single-chain Fv, an Fab, an Fab′, and an F(ab′)2.

According to still further features in the described preferredembodiments, the AMP and/or AMP-like molecule is selected from the groupconsisting of a defensin, a cathelicidin, a cationic peptide, ahydrophobic peptide, a human AMP and a human AMP-like molecule.

According to still further features in the described preferredembodiments, the AMP is any one of the cathelicidin and/or cathelicidinfragments listed below as SEQ. ID NOS. 1-59.

According to still further features in the described preferredembodiments, the cell and/or tissue is selected from the groupconsisting of an synovial cell and/or tissue, a nerve cell and/ortissue, a beta cell and/or tissue, an osteoblast, osteocyte orosteoclast cell and/or tissue and an endothelial cell and/or tissue.

According to still further features in the described preferredembodiments, the biological process is selected from the groupconsisting of growth, differentiation, and associated with aninflammatory disease or autoimmunity.

According to a further aspect of the present invention there is provideda method of treating a disease in a subject, such as a mammal, forexample, a human patient, in need thereof, the method comprisingproviding to the subject a therapeutically effective amount of anantimicrobial peptide (AMP) and/or AMP-like molecule (and in particulara cathelicidin, active fragment thereof or active cathelicidin analog ofthe cathelicidin or the fragment thereof), thereby treating the diseasein the subject in need thereof.

According to further features in preferred embodiments of the inventiondescribed below, administering the AMP and/or AMP-like molecule to thesubject is effected by exposing a location of the subject to a carrierwhich includes the AMP and/or AMP-like molecule at a concentrationselected from a range of about 2 nanograms per milliliter to about 10micrograms per milliliter.

According to still further features in the described preferredembodiments, administering the AMP and/or AMP-like molecule to thesubject is effected via a route selected from the group consisting ofthe topical, intranasal, transdermal, intradermal, oral, buccal,intravenous, parenteral, rectal and inhalation route.

According to still further features in the described preferredembodiments, the subject is human.

According to yet a further aspect of the present invention there isprovided an article of manufacture comprising packaging material and apharmaceutical composition, the article of manufacture being identifiedfor treatment of a disease being associated with a biological process ina cell and/or tissue, the biological process being selected from thegroup consisting of growth, differentiation, or inflammation associatedwith a disease; the pharmaceutical composition comprising apharmaceutically acceptable carrier and, as an active ingredient, anantimicrobial peptide (AMP) and/or AMP-like molecule.

According to further features in preferred embodiments of the inventiondescribed below, the pharmaceutically acceptable carrier is selected soas to enable administration of the pharmaceutical composition via aroute selected from the group consisting of the topical, intranasal,transdermal, intravenous, intradermal, oral, buccal, parenteral, rectaland inhalation route. The pharmaceutically acceptable carrier may, forexample, be of the sort of carriers known in the art for the delivery oftherapeutic peptides. The pharmaceutically acceptable carrier may, forexample, be other than water alone or other than water altogether.

According to still further features in the described preferredembodiments, the pharmaceutical composition is formulated as a solution,suspension, emulsion or gel.

According to still further features in the described preferredembodiments, the pharmaceutical composition is composed so as to enableexposure of a cell and/or tissue of a subject having the disease to thecompound at a concentration selected from a range of about 2 nanogramsper milliliter to about 10 micrograms per milliliter.

According to a further aspect of the present invention there is provideda method of treating an autoimmune disease, chronic inflammatorydisease, an inflammatory disease, a cancer, the method comprising ofdelivering the AMP or analog thereof, in particular a cathelicidin AMPto a human subject or mammal, thereby regulating the biological processin the subject.

According to still a further aspect of the present invention there isprovided a method of regulating a biological process in a cell and/ortissue, the method comprising exposing the cell and/or tissue to anantimicrobial peptide (AMP) and/or AMP-like molecule, thereby regulatingthe biological process in the cell and/or tissue.

According to further features in preferred embodiments of the inventiondescribed below, exposing the cell and/or tissue to the AMP and/orAMP-like molecule is effected by providing the AMP and/or AMP-likemolecule to a subject.

According to still further features in the described preferredembodiments, the providing to the subject the AMP and/or AMP-likemolecule is effected by administering the AMP and/or AMP-like moleculeto the subject and/or by expressing the AMP and/or AMP-like molecule inthe subject.

According to still further features in the described preferredembodiments, the exposing the cell and/or tissue to the AMP and/orAMP-like molecule is effected by exposing the cell and/or tissue to theAMP and/or AMP-like molecule at a concentration selected from a range ofabout 2 nanograms per milliliter to about 10 micrograms per milliliteror from about 10 micrograms per milliliter to about 30 micrograms permilliliter.

According to still further features in the described preferredembodiments, the AMP and/or AMP-like molecule is selected from the groupconsisting of and LL-37 or analogs of LL-37 or other cathelicidins andcathelicidin fragments or analogs as listed below.

According to still further features in the described preferredembodiments, the cell and/or tissue is derived from a human.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing: (i) a method of treating adisease which is associated with a biological process in a cell/tissuesuch as growth, differentiation, inflammation, metastasis and/orangiogenesis by using a compound which is capable of regulatinglevels/activity of an AMP and/or an AMP-like molecule, of decreasingolevels/activity of an AMP and/or an AMP-like molecule; and/or by usingan AMP and/or an AMP-like molecule or by increasing levels/activity ofan AMP and/or an AMP-like molecule; (ii) an article of manufactureincluding such a compound and being labeled for treatment of such adisease; and (iii) a method of identifying such a compound.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

FIG. 1 is a graph depicting incidence of arthritis in mouse model ofcollagen induced arthritis. Treatment using cathelicidin 34a.a. mCRAMPpeptide (experimental group) at a concentration of 1.5 mg/kg.Subsequently on days 2 and 4 post immunization, the dose was reduced to1.0 mg/kg. Starting with day 7 and through day 72, a dose of 0.8 mg/kgwas used. All treatments were performed 3 times per week, on a Monday,Wednesday, and Friday schedule, and the peptide or control vehicle wasadministered intraperitoneally for each treatment, rotating injectionareas. All mice were weighed at the beginning of the experiment in orderto calculate dosage administered. At day 49, the mice were again weighed(average of 1.6 gm increase) and dosages were adjusted accordingly.

Starting on day 11, all mice were examined 3 times per week forincidence and severity of arthritis and each arthritic limb was assigneda numerical score based on the degree of inflammation observed accordingto the scale below.

Erythema or mild swelling to the tarsals, metatarsal, foot, digits,ankylosis or ankle joint in any one of the four legs marks incidence ofarthritis. As can be seen, incidence rate is greater in the controlgroup.

Further analysis of incidence rate of inflamed paws in all mice is shownin FIG. 6.

The sequence of mCRAMP is: gllrkggekigeklkkigqkiknffqklvpqpeq (SEQ NO61).

FIG. 2 is a graph depicting Severity of Arthritis—The severity ofarthritis was analyzed on the basis of degree of inflammation scored asfollows and the number of affected limbs. 0—No evidence of erythema andswelling, 1—Erythema & mild swelling confined to the tarsals or anklejoint, 2—Erythema & mild swelling extending from the ankle to thetarsals, 3—Erythema & moderate swelling extending from the ankle tometatarsal joints, 4—Erythema & severe swelling encompass the ankle,foot, and digits, or ankylosis of the joint.

As seen in the FIG. 2, differences between the two groups were clearlyobserved when analyzed as mean Severity Score/Mouse. While these dataare weighted somewhat by the differences in arthritis incidence, thedifferences in the severity appear to be even greater than thedifferences in incidence. Data in FIG. 2 is from the same experimentdescribed in FIG. 1.

FIG. 3 is a graph depicting the number of Arthritic Limbs/Mouse. Similarto the Severity/Mouse score as in FIG. 2, the number of ArthriticLimbs/Mouse was also generally lower in the experimental group, althoughthe appearance of arthritic limbs followed similar kinetics as thecontrol group, but at a delayed incidence.

Data in FIG. 3 is from the same experiment described in FIG. 1 and FIG.2.

FIG. 4 is a graph showing a follow-up of clinical score from the day ofincidence of arthritis until day 19 after incidence. This follow-up isrequired since each arthritic mouse develops an incidence ofinflammation on any one of four paws at a varying number of days sincethe beginning of the experiment. Therefore in order to determine thesignificance level between the groups it is necessary to run a follow-uptest statistic. Data in FIG. 4 is from the same experiment described inFIG. 1 and FIG. 2.

FIG. 5 is a graph showing the sum of the severity index of clinicalscore in all mice of control versus treatment group during the time indays since immunization.

A clear trend is shown of greater severity of disease in the controlgroup from day 27 onwards. Data in FIG. 5 is from the same experimentdescribed in FIG. 1 and FIG. 2.

FIG. 6 is a graph showing the incidence of arthritic paws in treatmentversus control groups. Any one mouse may be included in this data up tofour times corresponding to four different paws in any one mouse. Atrend line is computed for each of the treatment and control groupsusing Microsoft excel technology. Data in FIG. 6 is from the sameexperiment described in FIG. 1 and FIG. 2.

FIG. 7 shows a table listing the results of the mouse ExperimentalAutoimmune Encephalitis (EAE) model.

C57BL/6 (B6) mice were purchased from Harlan (Jerusalem, Israel).Female, 9 week old mice were used in the experiment. The mice werehoused in the specific-pathogen free (SPF) animal facility of the HebrewUniversity and all experiments were approved by the institutional animalcare and use committee (IACUC).

MOGB35-55B peptide (MEVGWYRSPFSRVVHLYRNGK) 1.25 mg/ml in PBS wasemulsified in complete Freund's adjuvant (CFA) supplemented with 400 μgM. tuberculosis (Mt) H37RA (Difco). Mice were immunized s.c. in theflank with 250 μg MOGB35-55B/CFA using a 25G needle. 200 ng PertussisToxin (Sigma) was injected i.v. at the time of immunization and 48 hlater. EAE was scored on a scale of 0-6: 0, no impairment; 1, limp tail;2, limp tail and hind limb paresis; 3, ≧1 hind limb paralysis; 4, fullhind limb and hind body paralysis; 5, hind body paralysis and front limbparesis; 6, death. Mice were treated with the cathelicidin peptidesupplied by Biosight Ltd. Karmiel, Israel diluted in PBS, vs. PBS as acontrol. Cathelicidin (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) (SEQ NO 61)was diluted in sterile PBS and divided to aliquots kept at −20° C. suchthat each aliquot was thawed once for use. Mice were treated byintraperitoneal (i.p.) injection of roughly 200 ul volume (adjusted forweight) 3 times a week (Sun-Tues-Thurs) starting the day of immunizationwith MOG/CFA and through day 48. Clinical EAE scores were evaluatedthrough day 60. Dosage of Cathelicidin injections (IP) was 2 mg/Kg and0.2 mg/Kg. There were six mice in each group (total of 18 mice).

Of particular note is the fact that all the mice who developed EAEeventually died by day 50 while none of the mice in either of thetreatment groups died even by day 60.

There is a clear significant difference in average clinical score and inAverage score at first peak of disease.

The lower dose of peptide, 0.2 mg/Kg was more protective than the higher2 mg/Kg dose.

FIG. 8 shows a graph of the average clinical score for each day afterimmunization for the three groups in the EAE experiment as described inFIG. 7.

FIG. 9A, FIGS. 9B, and 9C shows photographs taken on day 60 of the threeremaining healthy mice in the control group, all six remaining live micein the low dose group, and two examples of EAE affected mice havingparalyzed hind legs and tail.

FIG. 10 shows a Western blot analysis of 4 different scFv developed thatbind LL-37.

FIG. 11 shows the inhibitory effect of scFv on LL-37 in bacteria killingassays. In order to find out the concentration of LL37 at which 50% ofthe bacteria could be killed (called “IC50”). Basically the activityprotocol follows the ability of the antibody to block the antimicrobialactivity of LL-37. The bacteria used were Pseudomonas that was isolatedfrom a wound. The growth medium was LB. LL-37 was added at aconcentration of 100 microgram/ml (the final volume or the reaction is50 microliter). Blocking antibodies at 1 or 5 microliter of antibody(=1:50 or 1:10 dilutions respectively. Low antibody levels ensure anon-specific effect. Concentration of bacteria was estimated by opticaldensity (OD) reading at 490.

FIG. 12 shows the percentage change in glucose levels two hoursfollowing an LPS injection in treatment vs. control. LPS wasadministered to C57BL/6 mice at 0.2 mg/kg. Mice were bled approximately2 h after LPS injection (T=0). Changes in glucose levels were examined.

FIG. 13 shows the average weight gain in male DBA/1 mice about ten weeksof age being fed on a normal no-high fat diet for 21 days. Two groups ofmice, 10 in each group were weighed. The control increased in weight atan average of 0.0536 gms per day whereas the treatment (cathelicidinmCRAMP at 0.8 ug/ml) increased at 0.0488 per day.

FIG. 14 shows a similar experiment as in FIG. 13 only that the mice weregiven 0.4 ug/ml and were weighed 3 times a week while being fed a highfat diet of 60% Kcal.

FIG. 15A and FIG. 15B shows a list of mouse paws selected for analysisfor bone resortion, deformation, immunohistology and osteoclast analysisand counting. Mouse paws were obtained from experiment in example 1.

FIG. 16, FIG. 17, FIG. 18 and FIG. 19 shows the histology analysisshowing a beneficial effect of cathelicidin on bone with reduced boneresorption in treatment group even the inflamed treatment group had lessosteoclast than the non-inflamed control. Staining was done with H&E andfor tartrate-resistant acid phosphatase (TRAP).

FIG. 20 shows the effect of human beta defensin 2 given for a durationof 7 weeks on human psoriatic skin Inhibition by dominant negativepeptide analogues or fragments is suggested as a viable mode oftreatment for this disease

FIG. 21 shows a histogram depicting significant BETATC beta cell lineproliferation brought about by cathelicidin LL-37. Murine beta cell linewere treated for 3.5 days with LL-37 at 2 microgram/ml (red/dark bars)and compared to PBS control (red/dark bars). Cell proliferation wasestimated by measuring [3(H)]-thymidine incorporation and was expressedas percent of control untreated cells. A representative experiment isshown.

DETAILED DESCRIPTION

The present invention provides methods of using compounds capable ofincreasing activities/levels of antimicrobial peptides(AMP)/antimicrobial peptide-like molecules (AMLs) and/or of decreasingactivities/levels of antimicrobial peptides, (AMP)/antimicrobialpeptide-like molecules (AMLs) and/or of using AMPs/AMLs or analogs orfragments thereof for regulating in cells/tissues biological processessuch as growth, differentiation, growth/differentiation balance, ofmethods of using such molecules for treating diseases associated withsuch biological processes and/or which are amenable to treatment viaregulation of such biological processes; for treating autoimmunity,inflammation, metastasis and angiogenesis; of articles of manufacturewhich include such molecules and which are labeled as being for use intreating such diseases; and of methods of identifying such compoundscapable of regulating or increasing or decreasing activities/levels ofAMPs/AMLs and/or of identifying such AMPs/AMLs. Specifically, thepresent invention can be used to optimally treat a vast range ofdiseases associated with such biological processes, includinginflammatory diseases/diseases associated with cellularproliferation/differentiation imbalance, autoimmune and inflammatorydiseases such as multiple sclerosis, arthritis, obesity, insulinresistance, osteoporosis, periodontitis, and other diseases associatedwith autoimmunity and/or cellular proliferation/differentiationimbalance.

Specifically the AMP involved is endogenous cathelicidin or its analogsor fragments thereof.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Diseases which are associated with autoimmunity, inflammation, anddysregulated cell/tissue proliferation/differentiation, dysregulatedcell/tissue proliferation/differentiation balance, include a multitudeof diseases which are of great medical and/or economic impact and forwhich no satisfactory treatment methods are available. While conceivingthe present invention, the present inventors have hypothesized thatAMPs/AMLs are involved in the pathogenesis of such diseases, and/or andhence that methods of regulating or increasing or decreasingactivities/levels and/or administering such molecules and in particular,cathelicidins or analogs or fragments of cathelicidin could be used fortreating such diseases.

The prior art approach relating to such methods involves the inventionof the current inventors in WO 2004-056307 incorporated by referenceherein.

The prior art approach, however, relates to autoimmune diseases ingeneral and provides in-vivo data on psoriasis showing that cathelicidinis indeed an immune regulator in psoriasis. The present inventiontherefore, a continuation in part to the previous invention of thecurrent inventors, shows in-vivo data for various specific diseasesassociated with autoimmunity, inflammation including low gradeinflammation found in metabolic diseases as well as bone celldifferentiation/proliferation leading to bone degradation.

While reducing the present invention to practice, it was also uncoveredthat cathelicidin and therefore AMPs could be used to significantlyregulate growth of cultured mouse beta-cells.

Hence, in sharp contrast to prior art techniques, the method accordingto the present invention enables use of compounds capable of regulatingby either increasing or decreasing levels/activity of cathelicidin or ofother AMPs/AMLs, and/or the use of such cathelicidins or analogs orfragments thereof or other AMPs/AMLs for regulating biological processessuch as growth, differentiation, inflammation, and for the treatment ofnumerous specific diseases such as for example type 1 diabetes and otherdiseases such as those which are associated with inflammation,dysregulated cell proliferation/differentiation, chronic inflammatorydiseases and autoimmune diseases.

Thus, the present invention provides a method of regulating a biologicalprocess in a cell and/or tissue associated with a disease. The method iseffected by exposing the cell and/or tissue to: an AMP and in particulara cathelicidin peptide, an analog of a cathelicidin peptide, an analogof a cathelicidin peptide that has been designed to be more stablein-vivo so as not to break down into pro-inflammatory fragments ofitself, a cathelicidin analog functioning as a dominant negative or acathelicidin peptide that competes on binding to cognate receptors withan AMP without inducing disease, a compound being capable of decreasingor increasing an activity and/or level of an antimicrobial peptide (AMP)and/or AMP-like molecule (AML).

The method can be used to regulate in a cell/tissue a biological processsuch as growth, differentiation, autoimmunity and inflammation. Byvirtue of enabling regulation of such a biological process in acell/tissue, the method can be used for treating a disease which isassociated with such a biological process, and can be used foridentifying the regulator, as described in further detail herein below.Diseases associated with such biological processes include, for example,autoimmune diseases, diseases associated with dysregulated cell/tissuegrowth/proliferation balance, wound-associated diseases, and tumors.

As used herein, the term “regulator” refers to the compound which iscapable of decreasing an activity and/or level of an AMP/AML, increasingan activity and/or level of an AMP/AML, and/or to an AMP which is usedfor practicing any aspect of the present invention.

As used herein, the phrases “the compound”, “compound of the presentinvention”, and “AMP/AML inhibitor” interchangeably refer to thecompound which is capable of regulating, decreasing or increasing anactivity/level of an AMP/AML.

Any of various types of AMP/AML or AMP/AML inhibitors may be employedaccording to the teachings of the present invention for regulating thebiological process, depending on the application and purpose.

As used herein, the term “AMP” includes any cathelicidin, and/orincluding any naturally occurring variant of such a molecule, such as anatural mutant/polymorphic variant/allele of such a molecule, or anysynthetic variant of such a molecule.

As used herein, the term “AML” includes any molecule having a biologicalactivity which is substantially similar to that of a cathelicidin,includes any molecule which substantially promotes the biologicalactivity of a cathelicidin, and/or includes any molecule which issubstantially structurally homologous to a cathelicidin. In such case,homology implied may, for example, vary between 50% to 60%, 60% to 70%,70% to 80%, 80% to 90%, 90% to 99%, 90%-100%, or at least 95%.

The method may be effected using a single regulator of the presentinvention, or using any combination of multiple regulators of thepresent invention.

The AMP/AML inhibitor may be: a molecule capable of binding the AMP/AML;an enzyme capable of cleaving the AMP/AML; an siRNA molecule capable ofinducing degradation of an mRNA encoding the AMP/AML; a DNAzyme capableof cleaving an mRNA or DNA encoding the AMP/AML; an antisensepolynucleotide capable of hybridizing with an mRNA encoding the AMP/AML;a ribozyme capable of cleaving an mRNA encoding the AMP/AML; anon-functional analog of at least a functional portion of the AMP/AML; amolecule capable of inhibiting activation or ligand binding of theAMP/AML; and a triplex-forming oligonucleotide capable of hybridizingwith a DNA encoding the AMP/AML.

Ample guidance for obtaining and utilizing such AMP/AML inhibitors isprovided herein below and in the literature of the art (for example,refer to U.S. Patent Application Pub. No. 20030044907 which isincorporated herein by reference).

The AMP/AML inhibitor may be any small molecule, AMP/AML dominantnegative, or polypeptide that competes with the AMPs for cognate cellreceptors without inducing disease. For example, the AMP/AML inhibitormay be a topological analog of an AMP/AML that has been engineered toremain anti microbial yet lose its chemoattracting ability. Engineeringof disulfide bridges to dissect antimicrobial and chemotactic activitiesof AMPs/AMLs such as human beta-defensin-3 can be performed aspreviously described (Wu Z. et al., 2003. Proc. Natl. Acad. Sci. U.S.A.100:8880-5).

The term “dominant negative mutant” as used herein refers to apolypeptide or a nucleic acid coding region sequence which has beenchanged with regard to at least one position in the sequence, relativeto the corresponding wild type native version at a position whichchanges an amino acid residue position at an active site required forbiological and/or pharmacological activity of the native peptide.Accordingly, dominant negative mutants or fragments of the cathelicidinpeptide as listed below and contemplated herein include, but are notlimited to, polypeptide species which manifest any change (substitutionand/or deletion) with regard to at least one amino acid of the AMP orcathelicidin peptide. Dominant negative mutant embodiments of theinvention are moreover nucleic acids which encode peptides, as well asthe peptides themselves, which comprise fragments of the AMP or morespecifically of the cathelicidin hCAP-18 and are listed as in (SEQ. IDNOS: 1-59).

The AMP/AML inhibitor may be a synthetic antibody mimic in whichmultiple peptide loops are attached to a molecular scaffold (describedin U.S. Pat. No. 5,770,380).

Such an AMP/AML mimic can be obtained, for example, by moleculeimprinting. This technique may be performed by preparing a polymer bycross-linking a monomer around a “template molecule” (the AMP/AML). Thistemplate molecule is removed after the polymerization of the monomer andits size, shape and chemical functions are recorded in the polymer. Thesites of the removed template molecule are named “imprint sites”. Thesesites allow the recognition of the template molecule or close structuralmolecules. Molecularly imprinted polymers can serve as artificialbinding mimics as do natural antibodies.

The molecule capable of inhibiting activation or ligand binding of theAMP/AML may advantageously inhibit binding of a receptor expressed oncell, such as a leukocyte, which binds the AMP/AML to inhibit abiological process mediated by binding of the AMP/AML to the receptor.Examples of such AMPs/AMLs and cognate receptors thereof are shown inTable 1.

TABLE 1 AMPs/AMLs and cognate cell receptors, and diseases associatedwith interaction therebetween Receptor- AMP/AML Receptor expressingcells Disease LL-37 EGFR, FPRL1 Monocyte, Psoriasis, rheumatoiddendritic arthritis (RA), atopic cell, T cell, dermatitis, contactneutrophils, dermatitis, chronic eosinophils, hepatitis, inflammatoryleukocytes, bowel disease (IBD), epithelial cell, allergy, B cellendothelial malignancies, cells hepatocellular carcinoma, pancreaticadenocarcinoma and others beta- Toll l-like Dendritic defensin-2receptor- 4 cells beta- Toll-like defensin-2 receptor-2 betaCC-chemokine Hematopoietic Psoriasis, RA, atopic defensin-1 receptor-6cells, dermatitis, contact beta (CCR6) dendritic dermatitis, chronicdefensin-2 cells, hepatitis, IBD, allergy, B cell malignancies,hepatocellular carcinoma, pancreatic adenocarcinoma and more defensin-5Intestinal Crohn's disease mucosa adreno- L1 and gastric IBD, allergy,medullin calcitonin epithelial hepatocellular receptor-like cellscarcinoma, and more receptor (CRLR)

Of particular note is that cathelicidin antimicrobial peptides blockdendritic cell TLR4 activation (J Immunol. 2007 Feb. 1; 178(3):1829-34)and therefore cathelicidins would act as inhibitors to beta-defensinactivation of TLR4.

Further examples of receptors of AMPs/AMLs such as chemokines, the cellsin which such receptors are expressed, and the diseases in which theinteraction between such AMPs/AMLs and such receptors are involved areprovided in D'Ambrosio et al., 2003. J. Immunol. Methods 273 3-13.

The activity of LL-37 (Weiner, D J. et al., 2003. Am. J. Respir. CellMol. Biol. 28:738-745), defensin-3, lactoferrin and IL-8 (Perks, B. etal., 2000. Am. J. Respir. Crit Care Med. 162:1767-1772) is inhibited byF-actin, further inhibitors therefore the AMP/AML inhibitor may beF-actin. F-actin forms bundles in the presence of the polycationicinterleukin IL-8, therefore F-actin is an inhibitor of downstreamelements of the ligand-receptor connectivity of both LL-37 andinterleukin IL-8. LL-37 and defensin-3 are inhibited by gelsolin,therefore the AMP/AML inhibitor may be gelsolin. Serpins and theiranalogs or fragments are inactivators of AMP by formation of complexeswith AMP (Panyutich, A V. et al., 1995. Am. J. Respir. Cell Mol. Biol.12:351-357; alpha-1 antichymotrypsin, the antimicrobial proteins alphaPI, SLPI and elafin are serpins that form complexes with other AMPs)thereby reducing specific types of inflammation (Hiemstra, P S, 2002.Biochem. Soc. Trans. 30:116-120), therefore the AMP/AML inhibitor may beserpins and their analogs or fragments. The AMP/AML inhibitor may beSIC, a secreted protein of streptococcus pyogenes that inactivatesantibacterial peptides.

Other AMP inhibitors or specifically cathelicidin inhibitors include,Alpha 2-Macroglobulin-Proteinase Complexes (Patrik Nyberg et al THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 51, Issue of December 17,pp. 52820-52823, 2004), aureolysin production by S. aureus andtaphylococcus and aureus-Derived Proteinases (Antimicrobial agents andchemotherapy, December 2004, p. 4673-4679), Elastolytic Cathepsins(Journal of Immunology, 2003, 170: 931-937), SufA—a novelsubtilisin-like serine proteinase of Finegoldia magna (Microbiology(2007), 153, 4208-4218).

Preferably, the molecule capable of binding the AMP/AML is an antibodyor an antibody fragment.

Alternately, the molecule capable of binding the AMP/AML may be any ofvarious type of molecule, including non-immunoglobulin peptides andpolypeptides,

Preferably, the antibody fragment is selected from the group consistingof a single-chain Fv, an Fab, an Fab′, and an F(ab′)2.

As used herein, the term “antibody” refers to a substantially intactantibody molecule. The antibody may, for example, be an IgG, IgA or IgM.Antibodies used according to the invention may be monoclonal antibodiesor polyclonal antibodies. The antibodies may, for example, be non-human,human or humanized antibodies.

As used herein, the phrase “antibody fragment” refers to a functionalfragment of an antibody that is capable of binding to an AMP/AML.

Suitable antibody fragments for practicing the present invention includea complementarity-determining region (CDR) of an immunoglobulin lightchain (referred to herein as “light chain”), a CDR of an immunoglobulinheavy chain (referred to herein as “heavy chain”), a variable region ofa light chain, a variable region of a heavy chain, a light chain, aheavy chain, an Fd fragment, and antibody fragments comprisingessentially whole variable regions of both light and heavy chains suchas an Fv, a single chain Fv, an Fab, an Fab′, and an F(ab′)₂.

Functional antibody fragments comprising whole or essentially wholevariable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of thevariable region of the light chain and the variable region of the heavychain expressed as two chains;

(ii) single chain Fv (“scFv”), a genetically engineered single chainmolecule including the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker.

(iii) Fab, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme papain to yield the intact lightchain and the Fd fragment of the heavy chain which consists of thevariable and C_(H)1 domains thereof;

(iv) Fab′, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin, followed by reduction(two Fab′ fragments are obtained per antibody molecule); and

(v) F(ab′)₂, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule which can be obtained bytreating whole antibody with the enzyme pepsin (i.e., a dimer of Fab′fragments held together by two disulfide bonds).

Methods of generating antibodies (i.e., monoclonal and polyclonal) arewell known in the art. Antibodies may be generated via any one ofseveral methods known in the art, which methods can employ induction ofin-vivo production of antibody molecules, screening of immunoglobulinlibraries (Orlandi D. R. et al., 1989. Proc. Natl. Acad. Sci. U.S.A.86:3833-3837; Winter G. et al., 1991. Nature 349:293-299) or generationof monoclonal antibody molecules by continuous cell lines in culture.These include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the Epstein-Barr virus(EBV)-hybridoma technique (Kohler G. et al., 1975. Nature 256:495-497;Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42; Cote R J. et al.,1983. Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030; Cole S P. et al.,1984. Mol. Cell. Biol. 62:109-120).

In cases where target antigens are too small to elicit an adequateimmunogenic response when generating antibodies in-vivo, such antigens(haptens) can be coupled to antigenically neutral carriers such askeyhole limpet hemocyanin (KLH) or serum albumin [e.g., bovine serumalbumin (BSA)] carriers (see, for example, U.S. Pat. Nos. 5,189,178 and5,239,078]. Coupling a hapten to a carrier can be effected using methodswell known in the art. For example, direct coupling to amino groups canbe effected and optionally followed by reduction of the imino linkageformed. Alternatively, the carrier can be coupled using condensingagents such as dicyclohexyl carbodiimide or other carbodiimidedehydrating agents. Linker compounds can also be used to effect thecoupling; both homobifunctional and heterobifunctional linkers areavailable from Pierce Chemical Company, Rockford, Ill. The resultingimmunogenic complex can then be injected into suitable mammaliansubjects such as mice, rabbits, and the like. Suitable protocols involverepeated injection of the immunogen in the presence of adjuvantsaccording to a schedule which boosts production of antibodies in theserum. The titers of the immune serum can readily be measured usingimmunoassay procedures which are well known in the art.

The antisera obtained can be used directly or monoclonal antibodies maybe obtained as described hereinabove.

Antibody fragments can be obtained using methods well known in the art.[(see, for example, Harlow and Lane, “Antibodies: A Laboratory Manual”,Cold Spring Harbor Laboratory, New York, (1988)]. For example, antibodyfragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g., Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Alternatively, antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. As describedhereinabove, an (Fab′)₂ antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages to produce 3.5S Fab′ monovalentfragments. Alternatively, enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. Ample guidancefor practicing such methods is provided in the literature of the art(for example, refer to: Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647; Porter, R R., 1959. Biochem. J. 73:119-126). Other methods ofcleaving antibodies, such as separation of heavy chains to formmonovalent light-heavy chain fragments, further cleavage of fragments,or other enzymatic, chemical, or genetic techniques may also be used, solong as the fragments bind to the antigen that is recognized by theintact antibody.

As described hereinabove, an Fv is composed of paired heavy chainvariable and light chain variable domains. This association may benoncovalent (see, for example, Inbar et al., 1972. Proc. Natl. Acad.Sci. USA. 69:2659-62). Alternatively, as described hereinabove, thevariable domains can be linked to generate a single chain Fv by anintermolecular disulfide bond, or alternately, such chains may becross-linked by chemicals such as glutaraldehyde.

Preferably, the Fv is a single chain Fv.

Single chain Fv's are prepared by constructing a structural genecomprising DNA sequences encoding the heavy chain variable and lightchain variable domains connected by an oligonucleotide encoding apeptide linker. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two variable domains. Ample guidancefor producing single chain Fv's is provided in the literature of the art(for example, refer to: Whitlow and Filpula, 1991. Methods 2:97-105;Bird et al., 1988. Science 242:423-426; Pack et al., 1993.Bio/Technology 11:1271-77; and Ladner et al., U.S. Pat. No. 4,946,778).

Isolated complementarity determining region peptides can be obtained byconstructing genes encoding the complementarity determining region of anantibody of interest. Such genes may be prepared, for example, by RT-PCRof mRNA of an antibody-producing cell. Ample guidance for practicingsuch methods is provided in the literature of the art (for example,refer to Larrick and Fry, 1991. Methods 2:106-10).

It will be appreciated that for human therapy or diagnostics, humanizedantibodies are preferably used. Humanized forms of non human (e.g.,murine) antibodies are genetically engineered chimeric antibodies orantibody fragments having—preferably minimal—portions derived from nonhuman antibodies. Humanized antibodies include antibodies in whichcomplementary determining regions of a human antibody (recipientantibody) are replaced by residues from a complementarity determiningregion of a non human species (donor antibody) such as mouse, rat orrabbit having the desired functionality. In some instances, Fv frameworkresidues of the human antibody are replaced by corresponding non humanresidues. Humanized antibodies may also comprise residues which arefound neither in the recipient antibody nor in the importedcomplementarity determining region or framework sequences. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the complementarity determining regions correspond to those of a nonhuman antibody and all, or substantially all, of the framework regionscorrespond to those of a relevant human consensus sequence. Humanizedantibodies optimally also include at least a portion of an antibodyconstant region, such as an Fc region, typically derived from a humanantibody (see, for example, Jones et al., 1986. Nature 321:522-525;Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr. Op.Struct. Biol. 2:593-596).

Methods for humanizing non human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non human. These non humanamino acid residues are often referred to as imported residues which aretypically taken from an imported variable domain. Humanization can beessentially performed as described (see, for example: Jones et al.,1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988. Science 239:1534-1536; U.S. Pat. No. 4,816,567)by substituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanized antibodies are chimeric antibodies, wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non human species. In practice, humanizedantibodies may be typically human antibodies in which somecomplementarity determining region residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies.

Thus, for example, antibodies used in the treatments of the inventionmay be humanized antibodies against LL-37 or against an epitope ofhCAP-18.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [see, for example, Hoogenboomand Winter, 1991. J. Mol. Biol. 227:381; Marks et al., 1991. J. Mol.Biol. 222:581; Cole et al., “Monoclonal Antibodies and Cancer Therapy”,Alan R. Liss, pp. 77 (1985); Boerner et al., 1991. J. Immunol.147:86-95). Humanized antibodies can also be made by introducingsequences encoding human immunoglobulin loci into transgenic animals,e.g., into mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon antigenic challenge, humanantibody production is observed in such animals which closely resemblesthat seen in humans in all respects, including gene rearrangement, chainassembly, and antibody repertoire. Ample guidance for practicing such anapproach is provided in the literature of the art (for example, referto: U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126,5,633,425, and 5,661,016; Marks et al., 1992. Bio/Technology 10:779-783;Lonberg et al., 1994. Nature 368:856-859; Morrison, 1994. Nature368:812-13; Fishwild et al., 1996. Nature Biotechnology 14:845-51;Neuberger, 1996. Nature Biotechnology 14:826; Lonberg and Huszar, 1995.Intern. Rev. Immunol. 13:65-93; Kellermann, S A. et al., 2002. Curr. Op.Biotechnol. 13:593-597).

Once antibodies are obtained, they may be tested for activity, forexample via ELISA.

Suitable antibodies may in many cases be purchased ready for use fromcommercial suppliers, such as Pharmingen, Dako, Becton-Dickinson,Sigma-Aldrich, and the like. Algae can be used to industriallymass-produce antibodies (Proc Natl Acad Sci USA. 2003, 100:438-42).

As described hereinabove, the AMP/AML inhibitor may be a smallinterfering RNA (siRNA) molecule. RNA interference is a two stepprocess. the first step, which is termed as the initiation step, inputdsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs(siRNA), probably by the action of Dicer, a member of the RNase IIIfamily of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA(introduced directly or via a transgene or a virus) in an ATP-dependentmanner. Successive cleavage events degrade the RNA to 19-21 bp duplexes(siRNA), each with 2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr.Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex tofrom the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen.2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12:225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the AMP/AML mRNA sequence is scanneddownstream of the AUG start codon for AA dinucleotide sequences.Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded aspotential siRNA target sites. Preferably, siRNA target sites areselected from the open reading frame, as untranslated regions (UTRs) arericher in regulatory protein binding sites. UTR-binding proteins and/ortranslation initiation complexes may interfere with binding of the siRNAendonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will beappreciated though, that siRNAs directed at untranslated regions mayalso be effective, as demonstrated for GAPDH wherein siRNA directed atthe 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA andcompletely abolished protein level(www.ambion.com/techlib/tn/91/912.html).

As used herein the term “about” refers to plus or minus 10%. Whereverthe term “about” occurs, it should be understood that the invention alsoprovides corresponding embodiments wherein the degree of variation isplus or minus 5%.

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

As described hereinabove, the AMP/AML inhibitor may be a DNAzymemolecule capable of specifically cleaving an mRNA transcript or DNAsequence of the AMP/AML. DNAzymes are single-stranded polynucleotideswhich are capable of cleaving both single and double stranded targetsequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997;943:4262). A general model (the “10-23” model) for the DNAzyme has beenproposed. “10-23” DNAzymes have a catalytic domain of 15deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions(Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for revof DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al.

As described hereinabove, the AMP/AML inhibitor may be an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding the AMP/AML.

Design of antisense molecules which can be used to efficiently decreaselevels/activity of an AMP/AML must be effected while considering twoaspects important to the antisense approach. The first aspect isdelivery of the oligonucleotide into the cytoplasm of the appropriatecells, while the second aspect is design of an oligonucleotide whichspecifically binds the designated mRNA within cells in a way whichinhibits translation thereof.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) andAoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat 130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)],while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients [GerwitzCurr Opin Mol Ther 1:297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60(2001)].

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

As described hereinabove, the AMP/AML inhibitor may be a ribozymemolecule capable of specifically cleaving an mRNA transcript encodingthe AMP/AML. Ribozymes are being increasingly used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest [Welch et al., Curr Opin Biotechnol.9:486-96 (1998)]. The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)].Most notably, several ribozyme gene therapy protocols for HIV patientsare already in Phase 1 trials. More recently, ribozymes have been usedfor transgenic animal research, gene target validation and pathwayelucidation. Several ribozymes are in various stages of clinical trials.ANGIOZYME was the first chemically synthesized ribozyme to be studied inhuman clinical trials. ANGIOZYME specifically inhibits formation of theVEGF-r (Vascular Endothelial Growth Factor receptor), a key component inthe angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well asother firms have demonstrated the importance of anti-angiogenesistherapeutics in animal models. HEPTAZYME, a ribozyme designed toselectively destroy Hepatitis C Virus (HCV) RNA, was found effective indecreasing Hepatitis C viral RNA in cell culture assays (RibozymePharmaceuticals, Incorporated—WEB home page).

As described hereinabove, the AMP/AML inhibitor may be a triplex formingoligonucleotides (TFOs). TFOs can be used for regulating the expressionof an AMP/AML gene in cells. Recent studies have shown that TFOs can bedesigned which can recognize and bind to polypurine/polypyrimidineregions in double-stranded helical DNA in a sequence-specific manner.These recognition rules are outlined by Maher III, L. J., et al.,Science, 1989; 245:725-730; Moser, H. E., et al., Science, 1987;238:645-630; Beal, P. A., et al, Science, 1992; 251:1360-1363; Cooney,M., et al., Science, 1988; 241:456-459; and Hogan, M. E., et al., EPPublication 375408. Modification of the oligonucleotides, such as theintroduction of intercalators and backbone substitutions, andoptimization of binding conditions (pH and cation concentration) haveaided in overcoming inherent obstacles to TFO activity such as chargerepulsion and instability, and it was recently shown that syntheticoligonucleotides can be targeted to specific sequences (for a recentreview see Seidman and Glazer, J Clin Invest 2003; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence: oligo, 3′--A G G T; duplex, 5′--A G C T; and duplex,3′--T C G A.

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sep. 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus for any given sequence in the AMP/AML gene a triplex formingsequence may be devised. Triplex-forming oligonucleotides preferably areat least 15, more preferably 25, still more preferably 30 or morenucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999;27:1176-81, and Puri, et al, J Biol Chem, 2001; 276:28991-98), and thesequence- and target specific downregulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31:833-43), and the pro-inflammatory ICAM-1gene (Besch et al, J Biol Chem, 2002; 277:32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 toEmanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

Techniques for administering such molecules to a cell or cellularstructure are routinely practiced by the ordinarily skilled artisan, andample guidance is provided in the literature of the art for suchadministration (refer, for example, to the references relevant to suchmolecules cited hereinabove and to U.S. Patent Application No.2003/0044907 which is incorporated herein by reference).

As described hereinabove, the method of regulating the biologicalprocess of the present invention comprises the step of exposing thecell/tissue to the regulator.

Exposing the cell/tissue to the regulator may be effected in variousways depending on the application and purpose. In cases where thecell/tissue form part of a human or an animal subject, exposing thecell/tissue to the regulator is preferably effected by providing theregulator to the subject.

Administering the regulator to a subject may be effected via anysuitable route facilitating exposure of the cell/tissue with theregulator, including a route selected from the group consisting of thetopical, intravenous, intranasal, transdermal, intradermal, oral,buccal, parenteral, rectal and inhalation route.

Preferably, subcutaneous and/or local injection of the regulator insaline solution is used for treating a disease such as arthritis.

Preferably, oral delivery in combination with aspirin or with NSAID ofthe regulator (such as for example cathelicidin or its analog orfragments or analogs of its fragments) in tablet form is used fortreating a disease such as arthritis or other inflammatory diseasesregularly treated by NSAID or aspirins such as for exampleatherosclerosis, osteoarthritis, or rheumatic diseases.

Preferably, topical application of the regulator in lipid or salinesolution, or in a cream on the skin is used for treating a cutaneousdisease such as a psoriasis legion.

Preferably, for treating respiratory diseases such as cystic fibrosisand asthma or COPD, the regulator is dissolved in a solution or providedin an inhalable powder form and administered using an inhaler.

Alternately, the cells may be exposed to regulator by expressing theregulator in the human or animal. In cases where the cell/tissue is acultured cell/tissue, exposing the regulator to the cell/tissue ispreferably effected by providing the regulator to the cell/tissuein-vitro using standard tissue culture methods. Preferably, providingthe regulator to the cell/tissue in-vitro is effected as described inthe Examples section which follows.

The regulator can be expressed in a subject by directly administering tothe subject a nucleic acid construct configured so as to suitablyexpress the regulator in-vivo. Alternatively, a nucleic acid constructfor expressing the regulator may be introduced into a suitable cellex-vivo via an appropriate gene delivery vehicle/method (transfection,transduction, homologous recombination, etc.), and using a suitablegenetic expression system as needed. The modified cells may be expandedin culture and administered to the subject where they will produce theregulator in-vivo. To enable cellular expression of the regulator, anucleic acid construct which encodes the regulator preferably includesat least one cis acting regulatory element, most preferably a promoterwhich is active in the specific cell population transformed. The nucleicacid construct can further include an enhancer, which can be adjacent ordistant to the promoter sequence and can function in up regulating thetranscription there from.

Suitable in vivo nucleic acid transfer techniques include transfectionwith viral or non-viral constructs, such as adenovirus, lentivirus,Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-basedsystems, polylysine based systems and dendrimers. Useful lipids forlipid-mediated transfer of the gene are, for example, DOTMA, DOPE, andDC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].The most preferred constructs for use in gene therapy are viruses, mostpreferably adenoviruses, AAV, lentiviruses, or retroviruses. A viralconstruct such as a retroviral construct includes at least onetranscriptional promoter/enhancer or locus-defining element(s), or otherelements that control gene expression by other means such as alternatesplicing, nuclear RNA export, or post-translational modification ofmessenger. Such vector constructs also include a packaging signal, longterminal repeats (LTRs) or portions thereof, and positive and negativestrand primer binding sites appropriate to the virus used, unless it isalready present in the viral construct. The construct may include asignal that directs polyadenylation, as well as one or more restrictionsites and a translation termination sequence. By way of example, such aconstructs will typically include a 5′ LTR, a tRNA binding site, apackaging signal, an origin of second-strand DNA synthesis, and a 3′ LTRor a portion thereof.

The various aspects of the present invention may be practiced by using,increasing or by decreasing the activity/level, of any of various typesof AMPs/AMLs, depending on the application and purpose. For example inthe experimental data use of cathelicidin peptide or its analog or itsfragment is shown for treating disease.

Preferably, the AMP/AML is a cationic and/or hydrophobic peptide.

As used herein, the term “peptide” (with the exception of the term inthe context of the phrases “antimicrobial peptide” or“antimicrobial-like peptide”, refers to a polypeptide which is composedof less than 51 amino acid residue.

Alternatively, a cathelidin may be more that 51 a.a. such as for examplehCAP-18 which contains and includes the pro-region of the LL-37 peptide.

Preferably, the AMP/AML is a cathelicidin.

Preferably, the cathelicidin is LL-37.

Preferably, the AMP/AML is of human origin. Alternately, it may be ofnon-human origin, in which case it is preferably of mammalian origin.

Numerous examples of AMPs/AMLs which may be used, and/or whoseactivity/levels may be decreased, for practicing the various aspects ofthe present invention are described in further detail herein below.

The method may be practiced so as to regulate the biological process inany of various cells/tissues of the present invention.

The cell/tissue is preferably from bone, synovial fluid or beta cells.

The method may be used to regulate the biological process in any ofvarious types of cells/tissues involved in disease included in thepresent invention.

The method may be affected by exposing the cell/tissue to the regulatorat any of various concentrations, depending on the application andpurpose.

Preferably, when using an AMP/AML inhibitor of the present invention forregulating the biological process, exposing the cell/tissue to theAMP/AML inhibitor is effected by exposing the cell/tissue to the AMP/AMLinhibitor at a concentration selected from a range of about 50 nanogramsper milliliter to about one milligram per milliliter.

Exposing the cell/tissue to the AMP/AML inhibitor may advantageously beeffected, depending on the application and purpose, by exposing thecell/tissue to the AMP/AML inhibitor at a concentration selected from arange of about 50 ng/ml to about 100 micrograms/ml, from a range ofabout 100 micrograms/ml to about 200 micrograms/ml, from a range ofabout 200 micrograms/ml to about 300 micrograms/ml, from a range ofabout 300 micrograms/ml to about 400 micrograms/ml, from a range ofabout 400 micrograms/ml to about 500 micrograms/ml, from a range ofabout 500 micrograms/ml to about 600 micrograms/ml, from a range ofabout 600 micrograms/ml to about 700 micrograms/ml, from a range ofabout 700 micrograms/ml to about 800 micrograms/ml, from a range ofabout 800 micrograms/ml to about 900 micrograms/ml, and from a range ofabout 900 micrograms/ml to about 1 mg/ml.

Preferably, when using an AMP/AML of the present invention forregulating the biological process, exposing the cell/tissue to theAMP/AML is effected by exposing the cell/tissue to the AMP/AML at aconcentration selected from a range of about 2 ng/ml to about 50micrograms/ml or from 50 micrograms/ml to 100 micrograms/ml. Exposingthe cell/tissue to the AMP/AML and in particular to cathelicidin mayadvantageously be effected, depending on inhibitor at a concentrationselected from a range of about 2 ng/ml to about 1 micrograms/ml, from arange of about 1 micrograms/ml to about 2 micrograms/ml, from a range ofabout 2 micrograms/ml to about 3 micrograms/ml, from a range of about 3micrograms/ml to about 4 micrograms/ml, from a range of about 4micrograms/ml to about 5 micrograms/ml, from a range of about 5micrograms/ml to about 6 micrograms/ml, from a range of about 6micrograms/ml to about 7 micrograms/ml, from a range of about 7micrograms/ml to about 8 micrograms/ml, from a range of about 8micrograms/ml to about 9 micrograms/ml, from a range of about 9micrograms/ml to about 10 mg/ml, from a range of about 10 micrograms/mlto about 11 micrograms/ml, from a range of about 11 micrograms/ml toabout 12 mg/ml, from a range of about 12 micrograms/ml to about 13micrograms/ml, from a range of about 13 micrograms/ml to about 17 mg/ml,from a range of about 17 micrograms/ml to about 20 micrograms/ml, from arange of about 20 micrograms/ml to about 25 mg/ml.

The method can be used to regulate in the cell/tissue a biologicalprocess such as growth, differentiation, inflammation or autoimmunity.

For regulating growth in an epithelial, skin and/or gastrointestinalcell/tissue, the regulator may advantageously be an AMP/AML inhibitor ofthe present invention and/or an AMP/AML (in particular, a cathelicidinor a cathelicidin analog) of the present invention.

As used herein, the phrase “cathelicidin inhibitor” refers to a compoundof the present invention which is capable of decreasing an activityand/or level of a cathelicidin.

As described hereinabove, the present invention can be used forregulating biological processes such as growth, differentiation,inflammation, chronic inflammation and autoimmunity. It will beappreciated that such biological processes are associated with thepathogenesis of numerous diseases, and that regulation of suchbiological processes according to the teachings of the present inventioncan be used for treating such diseases.

As used herein, the term “disease” refers to any medical disease,disorder, condition, or syndrome, or to any undesired and/or abnormalphysiological morphological, cosmetic and/or physical state and/orcondition.

Herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease,substantially ameliorating clinical symptoms of a disease orsubstantially preventing the appearance of clinical symptoms of adisease.

The method can be used for treating any of various diseases.

In particular, the method can be used for treating any of variousdiseases which are associated with: (i) inflammation; (ii) dysregulationof growth/differentiation of a cell/tissue; (iv) dysregulation ofgrowth/differentiation balance in bone (v) dysregulation ofgrowth/differentiation balance in beta cell function and insulinproduction (iii) dysregulation of growth/differentiation balance of acell/tissue; and (iv) autoimmunity.

Examples of such diseases, and others, which are amenable to treatmentvia the present invention are listed hereinbelow.

One of ordinary skill in the art, such as a physician, most preferably aphysician specialized in the disease, will possess the necessaryexpertise for treating a disease according to the teachings of thepresent invention.

As used herein, the phrase “subject in need thereof” refers to a subjecthaving the disease.

Preferably, the subject is a mammal, most preferably a human.

By virtue of demonstrably enabling regulation of growth of a pancreaticbeta cell cell/tissue, the method described above for inducing orinhibiting such growth is particularly suitable for treating any ofvarious diseases associated with dysregulated/diminished growth ofinsulin producing cells, and hence can be used for treating any ofvarious diseases associated with insulin depletion. Such diseasesnotably include diabetes mellitus type 1 or insulin dependant diabetesor type 2 diabetes, and other metabolic diseases including low gradeinflammatory diseases. In addition, the method of cell growth regulationas required in the treatment of cancer is also demonstrated.

By virtue of demonstrably enabling inhibition of Experimental AutoimmuneEncephalitis (EAE) inflammation in an in-vivo mouse model, the methoddescribed above for inhibiting such inflammation is particularlysuitable for treating any of various diseases associated with suchinflammation. Such diseases notably include inflammatory or autoimmunediseases, such as neurodegenerative disease, central nervous systemdiseases, multiple sclerosis, Alzheimer's disease, Parkinson's disease,myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmuneneuropathy, Lambert-Eaton myasthenic syndrome, paraneoplasticneurological disease, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy,Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmuneneuropathy, acquired neuromyotonia, arthrogryposis multiplex, opticneuritis, spongiform encephalopathy, migraine, headache, clusterheadache, and stiff-man syndromeautoimmune diseases.

By virtue of demonstrably enabling inhibition of Collagen inducedarthritis (CIA) inflammation in an in-vivo mouse model for rheumaticdiseases including rheumatoid arthritis, the method described above forinhibiting such inflammation is particularly suitable for treating anyof various diseases associated with such inflammation or autoimmunityand particularly in rheumatic diseases. Such diseases notably includeinflammatory or autoimmune diseases, such as arthritis, rheumaticdiseases and connective tissue/inflammatory diseases include arthritis,rheumatoid arthritis, pyogenic arthritis, mixed connective tissuedisease, cholesteatoma, lupus, relapsing polychondritis, autoimmunemyositis, primary Sjogren's syndrome, smooth muscle autoimmune disease,myositis, tendinitis, a ligament inflammation, chondritis, a jointinflammation, a synovial inflammation, carpal tunnel syndrome,osteoarthritis, ankylosing spondylitis, a skeletal inflammation, anautoimmune ear disease, osteoporosis, fibromyalgia, periodontitis, andan autoimmune disease of the inner ear, Diseases diagnosed or managed bythe rheumatologist include, Rheumatic diseases such as systemic LupusErythematosus, scleroderma (systemic sclerosis), dermatomyositis,polymyositis, polymyalgia rheumatica, osteoarthritis, septic arthritis,sarcoidosis, gout, pseudogout spondyloarthropathies, ankylosingspondylitis, reactive arthritis, psoriatic arthropathy, enteropathicspondylitis, reactive arthropathy vasculitis, polyarteritis nodosa,Henoch-Schönlein purpura, serum sickness, Wegener's granulomatosis,giant cell arteritis, temporal arteritis, Takayasu's arteritis, Behçet'ssyndrome, Kawasaki's disease (mucocutaneous lymph node syndrome),Buerger's disease (thromboangiitis obliterans), Juvenile IdiopathicArthritis (JIA).

By virtue of demonstrably enabling inhibition of weight gain andnutritional and Metabolic Diseases and low grade inflammation in anin-vivo mouse model that uses a high fat diet and LPS injections (IP) inorder to induce disease, the method described above for inhibiting suchinflammation is particularly suitable for treating any of variousdiseases associated with such inflammation. Such diseases notablyinclude inflammatory or autoimmune diseases, such as obesity, diabetes,insulin resistance, type 2 diabetes, Phenylketonuria (PKU), Metabolicsyndrome, Sodium metabolism disorders, Calcium metabolism disorders,Hypercalcemia, Hypocalcemia, Potassium metabolism disorders,Hyperkalemia, Hypokalemia, Phosphate metabolism disorders, Magnesiummetabolism disorders, Acid-Base metabolism disorders, atherosclerosis,cardio vascular diseases including; Aneurysm, Angina, Arrhythmia,Atherosclerosis, Cardiomyopathy, Cerebrovascular Accident (Stroke),Cerebrovascular disease, Congenital heart disease, Congestive HeartFailure, Myocarditis, Valve Disease, Coronary Artery Disease, Dilatedcardiomyopathy, Diastolic dysfunction, Endocarditis, High Blood Pressure(Hypertension), Hypertrophic cardiomyopathy, Mitral valve prolapse,Myocardial infarction (Heart Attack), Venous Thromboembolism.

By virtue of demonstrably enabling prevention of insulin resistance andMetabolic Diseases and low grade inflammation in an in-vivo mouse modelthat uses LPS injections (IP) in order to induce disease, the methoddescribed above for inhibiting such inflammation is particularlysuitable for treating any of various diseases associated with suchinflammation. Such diseases notably include inflammatory or autoimmunediseases, such as Fatigue, Intestinal bloating, Sleepiness, Weight gain,Increased blood triglyceride levels, Increased blood pressure,depression, Hyperglycemia, hyperglycaemia, or high blood sugar, diabetesmellitus, type 1 diabetes, type 2 diabetes, Polycystic ovarian syndrome(PCOS), Hypertension, Dyslipidemia that includes high triglyceridelevels, glandular/inflammatory diseases that include type B insulinresistance, Schmidt's syndrome, Cushing's syndrome, thyrotoxicosis,benign prostatic hyperplasia, pancreatic disease, Hashimoto'sthyroiditis, idiopathic adrenal atrophy, Graves' disease, androgenicalopecia, thyroid disease, thyroiditis, spontaneous autoimmunethyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmuneanti-sperm infertility, autoimmune prostatitis, Addison's disease, andType I autoimmune polyglandular syndrome.

By virtue of demonstrably enabling prevention of synovial pathology andbone resorption and degradation or deformation and low gradeinflammation in an in-vivo mouse model, the method described above forinhibiting such inflammation is particularly suitable for treating anyof various diseases associated with such inflammation. Such diseasesnotably include inflammatory or autoimmune diseases, such asosteoporosis, Osteogenesis imperfecta, Paget's disease, Osteochondroma,Osteomalacia, Osteomyelitis, Osteopetroses, Renal Osteodystrophy,Unicameral Bone Spurs, Bone Tumor, Craniosynostosis, Enchondroma,Fibrous Dysplasia, Giant Cell Tumor of Bone, Infectious Arthritis,Osteomyelitis, Klippel-Feil Syndrome, Limb Length Discrepancy,Osteochondritis Dissecans, periodontitis, bone loss in periodontitis,connective tissue/inflammatory diseases which include arthritis,rheumatoid arthritis, pyogenic arthritis, mixed connective tissuedisease, cholesteatoma, relapsing polychondritis, autoimmune myositis,primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis,tendinitis, a ligament inflammation, chondritis, a joint inflammation, asynovial inflammation, carpal tunnel syndrome, osteoarthritis,ankylosing spondylitis, a skeletal inflammation, an autoimmune eardisease, osteoporosis, fibromyalgia, periodontitis, and an autoimmunedisease of the inner ear.

By virtue of demonstrably enabling improvement of psoriasis and/or skininflammation in an in-vivo model, the method described above forinhibiting such inflammation is particularly suitable for treating anyof various diseases associated with such inflammation or for woundhealing. Such diseases notably include inflammatory or autoimmunediseases, such as cutaneous/inflammatory diseases include psoriasis,rosacea, dandruff, pemphigus vulgaris, lichen planus, atopic dermatitis,excema, scleroderma, dermatomyositis, alopecia, blepharitis, skincarcinoma, melanoma, squamous cell carcinoma, acne vulgaris, erythematoxicum neonatorum, folliculitis, skin wrinkles, autoimmune bullous skindisease, bullous pemphigoid, pemphigus foliaceus, dermatitis, and drugeruption.

For treating the disease, the regulator such as for example an AMP/AMLor the cathelicidin peptide or cathelicidin peptide analog or acathelicidin protein or a cathelicidin dominant negative analog or acathelicidin fragment or inhibitor may be administered via any ofvarious suitable regimens.

Depending on the application and purpose, each inter dose interval ofthe plurality of doses may advantageously be selected from a range ofabout 2.4 hours to about 3 days, from a range of about 3 days to about 6days, from a range of about 6 days to about 9 days, from a range ofabout 9 days to about 12 days, from a range of about 12 days to about 15days, from a range of about 15 days to about 18 days, from a range ofabout 18 days to about 21 days, from a range of about 21 days to about24 days, from a range of about 24 days to about 27 days, or from a rangeof about 27 days to about 30 days.

Preferably, the inter dose interval of the plurality of doses is about 1day. This bearing in mind that the half-life of LL-37 peptide in bloodof humans is approximately 3.4 days during which the peptide is usuallyprotected from degradation by LDL and HDL. (Infection and immunity,November 1999, p. 6084-6089).

If administered orally, the cathelicidin analog or peptide can bedelivered to the gastro intestinal tract (GIT). Peptides are usuallydelivered by injection or infusion due to their limited bioavailabilityand stability when delivered by other routs. The major problems toovercome in the development of oral peptide delivery are enzymaticdegradation and denatuartion in the GIT environment and their poorpenetration through physiological barriers (E. C. Lavelle et al. Vaccine15 (1997), pp. 1070-1078). However, microencapsulation technologiesusing synthetic polyesters such as poly(L-lactide) (L.PLA) andcopolymers such as poly(D,L-lactide co-glycolide) (PLG), used for thedelivery of drugs to humans and are now being considered for thedelivery of oral vaccines and peptides (M. Manocha et al Vaccine 23(2005) (48-49), pp. 5599-5617). The objective is an oral formulationsfor the delivery of a peptidic agent that needs to be delivered and actin the GIT. This objective is achieved by encapsulation of the peptideinto nanoparticles made of PLA or other pharmaceutically acceptablecarriers. These nanoparticles will protect the peptide fromdeterioration in the GIT and will allow adhesion and penetration to theGI mucosal tissue and being released in its active form. Suchformulations should be in a nanometer scale where the peptide is fullyprotected when in the GI fluid but being able to release the peptidewithin the GI mucosal tissue after absorption.

Two types of systems has been developed, encapsulation in PLA basedpolymer using the Liposphere technology and stereointeraction of thepeptide with stereoregular PLA. The first system is based on theformation of PLA nanoparticles coated with phospholipids by emulsionevaporation method. The second system is based on a physical interactionbetween the peptide and a common biodegradable stereoregular PLA. Inboth methods, PLA based polymers are used. These polymers are FDAapproved for the delivery of drugs as well as peptides and proteins. Inthis technology, the peptide is dissolved in a safe solvent along withthe polymer (D-PLA) which upon mixing the solution, a precipitate of thepeptide-PLA sterocomplex is formed. The precipitate can be of nanometerscale and contain a high load of the peptide. The peptide is releasedfrom the streocomplex as a result of hydrolysis of the PLA chain thatintertwined along the peptide chain. This system has been investigatedextensively for the injectable delivery of insulin, somatostatin andLHRH for extended release of weeks after injection. (Macromol Biosci.2006 Dec. 8; 6(12):1019-25, J Control Release 2005 Oct. 20;107(3):474-83, Biomaterials. 2002 November; 23(22):4389-96, MacromolBiosci. 2006 Dec. 8; 6(12):977-90)

As is described in the examples section which follows, in vivo mousemodels show by implication that administering 3 doses per week of acathelicidin regulator of the present invention to the subject (IP) withan inter dose interval of about 2.5 days can be used for effectivelytreating a disease such as Collagen Induced arthritis or EAE or multiplesclerosis or obesity of insulin resistance in a human subject.

Disease treatment may be effected via polytherapy by administration ofthe regulator in conjunction with Vitamin D3, calcitriol analogs, orpeptide inhibitors such as protease inhibitors, the serpin serineproteinase inhibitory components (alpha-1 PI) and alpha-1antichymotrypsin (Panyutich, A V. et al., 1995. Am. J. Respir. Cell Mol.Biol. 12:351-357), BAPTA-AM (an intracellular Ca(2+) chelating agent),pertussis toxin and U-73122 (a phospholipase C inhibitor; Niyonsaba, F.et al., 2001. Eur. J. Immunol. 31:1066-1075), T-cell targeted therapies,monoclonal antibody against chemokine tumor necrosis factor and cytokinetargeted therapies, fibroblast growth factor inhibitors. For example,topical treatments may advantageously include cell proliferationregulators such as retinoid—vitamin A—analog which modulates or changesthe cellular differentiation of the epidermis. Such polytherapy may beeffected using anti-inflammatory drugs/treatments as a precautionarymeasure against relapse of psoriasis or other auto-immune disease. Suchdrugs/treatments include tazarotene, methotrexate, acitretin,bexarotene, ploralem, etretinate, corticosteroid creams and ointments,synthetic vitamin D3, IL-10, IL-4 and IL-1RA (receptor antagonist).

A cathelicidin inhibitor/vitamin D combination is particularly claimedas a treatment modality for cancer. This is because whereas vitamin Dpathway can skew cancer cells into a desired differentiating state,cathelicidin, which is also expressed via the vitamin D pathway, skewscancer cells into the undesired proliferative state. Thus the maximumdesired differentiating non-proliferating pathway is achieved.

To enable treatment of the disease, the regulator is preferably includedas an active ingredient in a pharmaceutical composition which includes asuitable carrier and which is suitably packaged and labeled fortreatment of the disease.

The regulator according to the present invention can be administered toa subject per se, or in a pharmaceutical composition where it is mixedwith suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of active ingredients to an organism.

Herein the term “active ingredients” refers to the regulator of thepresent invention accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered active ingredients. An adjuvant isincluded under these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transnasal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated as described above using PLA based polymers or can beformulated readily by combining the active ingredients withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the active ingredients and a suitable powder base such as lactose orstarch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions. Cream solutions can include any lipids ororganic alcohols or chemicals including for example benzyl alcohol,macrogol, hexylene glycol, carbomer, ascorbic acid, butylhydroxyainisole, butyl hydroxytoluene, disodium edentate, water,trometamol, poxoamer.

Alternatively, the active ingredients may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

It should be understood that by administering a peptide of the inventionit is meant administering a peptide of the invention or apharmaceutically acceptable salt thereof or other pharmaceuticallyacceptable form thereof.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (regulator of the present invention) effective toprevent, alleviate or ameliorate symptoms of a disorder (e.g., psoriasisor a carcinoma) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredients which are sufficient toachieve a desired therapeutic effect (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredients. Thepack may, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

Thus, the present invention provides an article of manufacture whichcomprises packaging material identified for treatment of the disease,and a pharmaceutical composition which includes a pharmaceuticallyacceptable carrier and, as an active ingredient, the regulator.

Preferably, the pharmaceutical composition is formulated as a solution,suspension, emulsion or gel.

Preferably, the pharmaceutically acceptable carrier is selected so as toenable administration of the pharmaceutical composition via a routeselected from the group consisting of the topical, intravenous,intranasal, transdermal, intradermal, oral, buccal, parenteral, rectaland inhalation route.

Preferably, the pharmaceutical composition is composed so as to enableexposure of an affected cell/tissue of the subject having the disease,to the regulator at a suitable concentration, as described hereinabove,for treating the disease.

Preferably, the pharmaceutical composition is further identified foradministration to the subject according to a suitable regimen, asdescribed hereinabove.

Thus, the present invention provides a method of identifying a compoundcapable of treating autoimmune or inflammatory diseases by using amethod of regulating the biological process in a cell/tissue involved indisease. The method is effected in a first step by exposing thecell/tissue to a test compound which is: a compound capable ofdecreasing or increasing an activity and/or level of an antimicrobialpeptide (AMP) and/or AMP-like molecule (AML); and/or which is itself theAMP and/or AML. In a second step, the method is effected by evaluating acapacity of the test compound to regulate the biological process in thecell and/or tissue. In particular, the method involves the regulation ofthe AMP cathelicidin or by using the cathelicidin peptide or its analogor fragments.

It will be appreciated that the method of identifying the compound canbe used for screening a plurality of compounds so as to identify acompound having a desired capacity for regulating a biological process.

The method is preferably used to identify a compound capable ofregulating a biological process as described hereinabove with respect tothe method of the present invention of regulating a biological process.

Preferably, the test compound is a regulator as described hereinabovewith respect to the method of the present invention of regulating abiological process.

The method is preferably used to identify a compound capable ofregulating the biological process in the cell/tissue as describedhereinabove with respect to the method of the present invention ofregulating a biological process, and as described in the Examplessection which follows. As is described hereinabove with respect to themethod of regulating the biological process, and in the Examples sectionwhich follows, the method is preferably employed for identifying acompound which is capable of: inducing growth in an epithelial, bonecells, osteoclasts or osteoblasts, nerve, synovial cell/tissue, betacell, skin, keratinocytic and/or gastrointestinal cell/tissue;inhibiting growth in a tumor, epithelial, skin, keratinocytic and/orbone cells, osteoclasts or osteoblasts, nerve, synovial cell/tissue,beta cell, gastrointestinal cell/tissue; inhibitingangiogenesis/endothelial cell/tissue growth; inhibiting metastasis in atumor cell/tissue; correcting dysregulated balance ofproliferation/differentiation in an epithelial, keratinocytic and/orskin cell/tissue; and/or inhibiting inflammation in an epithelial,keratinocytic an/or skin cell/tissue.

The identification method may advantageously be performed usinghigh-throughput methodology. Ample guidance for practicing relevanthigh-throughput methods is provided in the literature of the art (refer,for example, to U.S. Patent Application No. 20030044907).

The test compound may be exposed to the cell/tissue in any of variousways. Preferably, the test compound is exposed to the cell/tissuein-vitro as described in the Examples section which follows.Alternately, the test compound may be exposed to the cell/tissue byexposing the test compound to a cultured cell/tissue.

Preferably, the cell which produces the test compound is a B-cellhybridoma. Alternately, the cell which produces the test compound may beof any of various types, depending on the application and purpose.

It will be appreciated that a B-cell hybridoma is an antibody producingcell, and hence that exposing the cell/tissue to a B-cell hybridoma canbe used for identifying a B-cell hybridoma which expresses an antibodywhich is capable of regulating the biological process.

Exposing the cell/tissue to the test compound may be effected byproviding the test compound to a subject which includes the cell/tissue(in-vivo model). Preferably providing the test compound to the testsubject is effected as described hereinabove with respect to providingthe regulator to a subject.

The identification method may be effected by exposing the test compoundto: lesions of any of various diseases associated with bone resorption,CNS disease including multiple sclerosis, arthritis, fat cells of theobeseepithelial wounds included in the present invention; a lesion in ananimal model as included in the examples of this invention; or a lesionin a human having the associated disease; a human biopsy of a normal orpathological involved lesion maintained in an organotypic culturecontaining plasma and lymphocytes of patients suffering from the diseasehaving and not having polymorphism on AMPs or their genes and promotersor that are induced to express an AMP (and in particular a cathelicidin)using known technologies such as transfection (Graham F L Virology 52(2): 456-67m, Bacchetti S Proc Natl Acad Sci USA 74 (4): 1590-4); and/orto a cell/tissue of a disease in which the disease inductive isoformsare ApoE4 and the non inductive isoform is ApoE3.

The identification method may be effected by exposing the test compoundto a human lesion biopsy grafted onto an animal (xenograft model),whereby the biopsy is taken with informed consent. The biopsy may betransplanted onto an immunodeficient mouse (for example, NIRS-bg-nu-xidor BNX). For establishing such a xenograft model, PBMCs may be isolatedfrom the blood obtained from the biopsy donor and activated (forexample, using a superantigen), and the animals injected with theactivated PBMCs. Ample guidance for practicing the identification methodusing such animal models is provided in Examples 6-8 of the Examplessection below and in the literature of the art (refer, for example, toU.S. Patent Application No. 20030044907).

Evaluation of the regulation of the biological processes encompassed bythe identification method may be effected using any of various suitablemethods known to the ordinarily skilled artisan. Preferably, suchevaluation is performed, where relevant, as described in the Examplessection which follows.

Evaluating regulation of the biological process may be effected usingquantitative evaluation or lessional thickness when using an in-vivomodel, cell count or histological evaluation.

Preferably, data obtained from the evaluation is processed usingstatistical analysis and ANOVA for maximum informativity.

According to one embodiment, exemplified by Example 3, theidentification method may involve exposing the test compound to culturedmicrobes/bacteria and evaluating regulation of the biological process iseffected by measuring survival of the microbes/bacteria. This may beeffected by a colony-forming unit assay performed with Staphylococcusaureus (isolated from clinical sample), GAS (NZ131), and enteroinvasiveEscherichia coli 029 as described (Porter et al, 1997). Before analysis,the concentration of the bacteria in culture will be determined byplating different bacterial dilutions. The protocol may be performed asfollows. Cells are washed twice with 10 mM sodium phosphate buffer (20mM NaH₂PO₄.H₂O, 20 mM Na₂HPO₄.7H₂O) and diluted to a concentration of2,000,000 cells per milliliter (S. aureus, GAS) or 200,000 cells permilliliter (E. coli) in phosphate buffer. S. aureus and E. coli areincubated for 4 hours at 37 degrees centigrade with variousconcentrations of an AMP/AML in the presence of various concentrationsof the test compound to be examined, in 50 microliters of buffer in 96well round bottom tissue culture plates (Costar 3799, Corning inc., NY).GAS are incubated for 1 hour due to the poor ability of GAS to grow insuch buffers. After incubation, the cells are diluted from 10× to100,000×, and each of 20 ml of those solutions are plated in triplicateon tryptic soy broth (for S. aureus) and Todd Hewitt broth (for GAS andE. coli), and the mean number of colonies is determined. The number ofcfu per ml is calculated, and the blocking activity of the examined testcompounds to block the bactericidal activities of the AMP/AML will becalculated as follows: (cell survival after AMP/AML incubation)/(cellsurvival after incubation without AMP/AML)×100, which represents thepercentage of cells that are alive, as compared to those which are not(cell survival after AMP/AML+test compound incubation)/(cell survivalafter incubation with test compound alone)×100.

All compounds identified will be screened for one or all of thefollowing effects: their ability to inhibit or regulate theantimicrobial activity of the AMP (cathelicidin in particular) or towhich they were raised against; their ability to affect theproliferation or differentiation or other cellular processes of culturedcells of the affected target tissue, originally isolated from normal ordiseased individuals or models, for example HaCaT, primary human ormurine keratinocytes or fibroblasts for screening for psoriasis; nervecells, bone cells or osteoclasts or osteoblasts and the effects of theinhibitors on activation of the immune system.

Identified compounds may be further screened for their effects onorganotypic cocultures and animal models so as to identify inhibitorsthat will be able to effectively inhibit a desired biological effect orcombination of biological effects. This may include, where suitable,identifying compounds that will inhibit or regulate the effects ofAMPs/AMLs on proliferation/differentiation balance but which maintaintheir antibacterial/antimicrobial activity.

The test compound or regulator may be any of various type of molecule,such as a small synthetic/non-polypeptidic molecule.

The test compound or regulator may advantageously be a peptide, aprotein or a glycosylated protein.

Test compounds and regulators of the present invention of any of varioussuitable types may be obtained from a commercial chemical library suchas, for example, one held by a large chemical company such as Merck,Glaxo Welcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly,Novartis, Pharmacia UpJohn, and the like. Test compounds and regulatorsof the present invention of any of various suitable types may also beordered via the World Wide Web (Internet) via companies such asChemcyclopedia(http://www.mediabrains.com/client/chemcyclop/BG1/search.asp).Alternatively, test compounds and regulators of the present invention ofany of various suitable types may be synthesized de novo using standardchemical and/or biological synthesis techniques. Ample guidance forsynthesis of molecules suitable for use as test compounds or regulatorsof the present invention of any of various suitable types is provided inthe literature of the art. For biological synthesis of molecules, suchas polypeptides and nucleic acids, refer, for example to: Sambrook etal., infra; and associated references in the Examples section whichfollows. For guidance regarding chemical synthesis of molecules, refer,for example to the extensive guidelines provided by The AmericanChemical Society (http://www.chemistry.org/portal/Chemistry). One ofordinary skill in the art, such as, for example, a chemist, will possessthe required expertise for chemical synthesis of suitable testcompounds.

In designing small molecules capable of binding the AMP/AML, severalfeatures, such as structures of antibody, receptors, ligands, andrelevant biochemical and biological data may be considered. Suchfeatures may include de novo folding design using energy minimizationand molecular dynamics, and comparative modeling followed by energyminimization and molecular dynamics. These two approaches differ only indeveloping the trial or initial structures. The folding patterns arestudied using energy minimization and molecular dynamics.

Cathelicidins or cathelicidin peptides include the hCAP-18 pro-peptideand its following fragments and/or analogs of these fragments sequences(SEQ ID NOs: 1-59) listed in order as follows:

SEQ ID 1: fdiscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 2:discdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 3:iscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 4:scdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 5:cdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 6:dkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 7:kdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 8:dnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 9:nkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 10:krfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 11:rfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 12:fallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 13:allgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 14:llgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 15:lgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 16:gdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 17:dffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 18:ffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 19:frkskekigkefkrivqrikdflrnlvprtes SEQ ID 20:rkskekigkefkrivqrikdflrnlvprtes SEQ ID 21:kskekigkefkrivqrikdflrnlvprtes SEQ ID 22: skekigkefkrivqrikdflrnlvprtesSEQ ID 23: llgdffrkskekigkefkrivqrikdflrnlvprte SEQ ID 24:llgdffrkskekigkefkrivqrikdflrnlvprt SEQ ID 25:llgdffrkskekigkefkrivqrikdflrnlvpr SEQ ID 26:llgdffrkskekigkefkrivqrikdflrnlvp SEQ ID 27:llgdffrkskekigkefkrivqrikdflrnlv SEQ ID 28:llgdffrkskekigkefkrivqrikdflrnl SEQ ID 29:llgdffrkskekigkefkrivqrikdflrn SEQ ID 30: llgdffrkskekigkefkrivqrikdflrSEQ ID 31: llgdffrkskekigkefkrivqrikdfl SEQ ID 32:llgdffrkskekigkefkrivqrikdf SEQ ID 33: llgdffrkskekigkefkrivqrikdSEQ ID 34: llgdffrkskekigkefkrivqrik SEQ ID 35: llgdffrkskekigkefkrivqriSEQ ID 36: llgdffrkskekigkefkrivqr SEQ ID 37: llgdffrkskekigkefkrivqSEQ ID 38: llgdffrkskekigkefkriv SEQ ID 39: llgdffrkskekigkefkriSEQ ID 40: efkriv SEQ ID 41: kefkrivq SEQ ID 42: gkefkrivqr SEQ ID 43:igkefkrivqri SEQ ID 44: kigkefkrivqrik SEQ ID 45: ekigkefkrivqrikdSEQ ID 46: kekigkefkrivqrikdf SEQ ID 47: skekigkefkrivqrikdfl SEQ ID 48:skekigkefkrivqrikdflrnlvprtes SEQ ID 49: kskekigkefkrivqrikdflrSEQ ID 50: rkskekigkefkrivqrikdflrn SEQ ID 51:frkskekigkefkrivqrikdflrnl SEQ ID 52: ffrkskekigkefkrivqrikdflrnlvSEQ ID 53: dffrkskekigkefkrivqrikdflrnlvp SEQ ID 54:gdffrkskekigkefkrivqrikdflrnlvpr SEQ ID 55:lgdffrkskekigkefkrivqrikdflrnlvprt

In accordance with the instant invention, AMPs, compositions comprisingthe same, and methods of use thereof are provided. In a particularembodiment, the antimicrobial peptide has at least 90% homology withamino acid sequence fallgdffrksk.X₁ (SEQ ID NO: 56), wherein X₁ isselected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, or 16 amino acids. The amino acid sequence of thepeptides may also be in reverse orientation. In another embodiment, theantimicrobial peptide has at least 90% homology with amino acid sequenceX.sub.ligkefkrivq.sub.2 (SEQ ID NO: 57), wherein X₁ is 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids and X₂ is 0, 1, 2,3, 4, 5, 6, 7, or 8 amino acids. In yet another embodiment, theantimicrobial peptide has at least 90% homology with amino acid sequenceX₁ffrkskekigkX₂ (SEQ ID NO: 57), wherein X₁ is 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids and X₂ is 0, 1, 2, 3, 4,5, 6, 7, or 8 amino acids. In yet another embodiment, the antimicrobialpeptide has at least 90% homology with amino acid sequence X₁vqrikdflrnX₂ (SEQ ID NO: 58) where X₂ is 0, 1, 2, 3, 4, 5, 6, 7 amino acids and X₁is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or19 amino acids. In yet another embodiment, the antimicrobial peptide hasat least 90% homology with amino acid sequence X₁gkefkrivqrikdflrn X₂(SEQ ID NO: 59) where X₂ is 0, 1, 2, 3, 4, 5, 6, 7 amino acids and X₁ is0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19amino acids.

In another embodiment, the antimicrobial peptide has at least 60%, 70%,80%, 90% or 95% homology (or identity) with amino acids in cathelicidinpeptides from mammals as described in (Curr Issues Mol Biol. 2005 July;7(2):179-96) namely:

RL-37: (SEQ ID NO: 60) RLGNFFRKVKEKIGGGLKKVGQKIKDFLGNLVPRTASRhesus monkey, CAP18: (SEQ ID NO: 62)GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY Rabbit, CRAMP: (SEQ NO 61)GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE Mouse, rCRAMP: (SEQ ID NO: 63)GLVRKGGEKFGEKLRKIGQKIKEFFQKLALEIEQ rat, CAP11: (SEQ ID NO: 64)(GLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI)Guinea, Canine cathelicdin: (SEQ ID NO: 65)KKIDRLKELITTGGQKIGEKIRRIGQRIKDFFKNLQPREEKS, Bac5: (SEQ ID NO: 66)RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP-NH2 Cow, Bac7:(SEQ ID NO: 67)RRIRPRPPRLPRPRPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPLCow, BMAP-27: (SEQ ID NO: 68) GRFKRFRKKFKKLFKKLSPVIPLLHL-NH₂Cow, BMAP-28: (SEQ ID NO: 69) GGLRSLGRKILRAWKKYGPIIVPIIRI-NH₂Cow, BMAP-34: (SEQ ID NO: 70) GLFRRLRDSIRRGQQKILEKARRIGERIKDIFR-NH₂Cow, Indolicidin: (SEQ ID NO: 71) ILPWKWPWWPWRR-NH2 Cow, Dodecapeptide:(SEQ ID NO: 72) RLCRIVVIRVCR Cow , Water buffalo cath (SEQ ID NO: 73)GLPWILLRWLFFR-NH2 Water buffalo, OADode: (SEQ ID NO: 74) RYCRIIFLRVCRSheep, SMAP-29: (SEQ ID NO: 75) RGLRRLGRKIAHGVKKYGPTVLRIIRIA-NH2Sheep, SMAP-34: (SEQ ID NO: 76) GLFGRLRDSLQRGGQKILEKAERIWCKIKDIFR-NH2Sheep, OaBac5 (SEQ ID NO: 77)RFRPPIRRPPIRPPFRPPFRPPVRPPIRPPFRPPFRPPIGPFP-NH2 Sheep, OaBac6:(SEQ ID NO: 78) RRLRPRHQHFPSERPWPKPLPLPLPRPGPRPWPKPLPLPLPRPGLRPWPKPLSheep, OaBac7.5: (SEQ ID NO: 79)RRLRPRRPRLPRPRPRPRPRPRSLPLPRPQPRRIPRPILLPWRPPRPIPRPQIQPIPRWLSheep, OaBac11: (SEQ ID NO: 80)RRLRPRRPRLPRPRPRPRPRPRSLPLPRPKPRPIPRPLPLPRPRPKPIPRPLPLPRPRPRRIPRPLPLPRPRPRPIPRPLPLPQPQPSPIPRPL Sheep, ChBac5: (SEQ ID NO: 81)RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFP-NH2 Goat, eCATH-1:(SEQ ID NO: 82) KRFGRLAKSFLRMRILLPRRKILLAS, eCATH-2: (SEQ ID NO: 83)KRRHWFPLSFQEFLEQLRRFRDQLPFP Horse, eCATH-3 (SEQ ID NO: 84)KRFHSVGSLIQRHQQMIRDKSEATRHGIRIITRPKLLLAS, PR-39: (SEQ ID NO: 85)RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP-NH2 Pig, (SEQ ID NO: 86)AFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPWFPPPPPFRPPPFGPPRFP-NH₂ Pig, Prophenin-2: (SEQ ID NO: 87)AFPPPNVPGPRFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPWFPPPPPFRPPPFGPPRFP-NH₂ Pig, Protegrin-1: (SEQ ID NO: 88)RGGRLCYCRRRFCVCVGR-NH₂ Pig, (SEQ ID NO: 89) RGGRLCYCRRRFCICV-NH2Pig, Protegrin-3: (SEQ ID NO: 90) RGGGLCYCRRRFCVCVGR-NH₂Pig , Protegrin-4: (SEQ ID NO: 91) RGGRLCYCRGWICFCVGR-NH₂Pig, Protegrin-5: (SEQ ID NO: 92) RGGRLCYCRPRFCVCVGR-NH₂ Pig, PMAP-23:(SEQ ID NO: 93) RIIDLLWRVRRPQKPKFVTVWVR Pig, PMAP-36: (SEQ ID NO: 94)GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGC-NH₂ Pig, PMAP-37: (SEQ ID NO: 95)GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQS

chCATH-B1: (Proc Natl Acad Sci USA. 2007 Sep. 18; 104(38):15063-8)chicken,

Canine cathelicidin (K9CATH): (Dev Comp Immunol. 2007; 31(12):1278-96),

Fowlicidin-3: (FEBS J. 2007 January; 274(2):418-28.), (J Biol Chem. 2006Feb. 3; 281(5):2858-67), (Immunogenetics. 2004 June; 56(3):170-7.)chicken,

CMAP27: (Vet Immunol Immunopathol. 2005 Jul. 15; 106(3-4):321-7),

Fish (cathelicidin from Atlantic cod and Atlantic salmon) Maier V H etal. Mol Immunol. 2008 Jul. 7.

Peptides with enhanced LPS neutralization and reduced pro-inflammatoryactivity are also included. Such peptides, for example 18-mer LLKKK orGKE and P60, P60.4, P60.4-Ac, CAP11 (cationic antibacterial polypeptideof 11 kDa), CAP18, GSLL-39, SMAP-29, and others as well as methods ofdiscovering such peptides are disclosed in CLINICAL AND DIAGNOSTICLABORATORY IMMUNOLOGY September 2002, p. 972-982 (18-mer LLKKK),ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, September 2006, p. 2983-2989 Vol.50, No. 9 (GKE), Laryngoscope. 2008 May; 118(5):816-20, Inflamm Res.2004 November; 53(11):609-22, Antimicrob Agents Chemother. 2005 July;49(7):2845-50, Protein Expr Purif. 2004 September; 37(1):229-35, Int JAntimicrob Agents. 2004 June; 23(6):606-12, Am J Respir Crit Care Med.2004 Jan. 15; 169(2):187-94, Eur J Biochem. 2002 February;269(4):1181-9, Surgery. 1995 June; 117(6):656-62, Prog Clin Biol Res.1995; 392:317-26, peptides 27(2006) 649-660 (P60, P60.4, P60.4-Ac), andP18 as in Biotechnol Lett (2008) 30:1183-1187 and are all incorporatedby reference herein. In particularly, such peptides having LPSneutralizing activity are included for the treatment of metabolicdiseases, obesity and insulin resistance as is demonstrated withcathelicidin in example 4 below.

Treatment using analogs of these above peptides, peptide fragments andproteins can be formed by modification as described below so as to makethe analog (modified cathelicidin peptide or modified peptide fragment)more stable in blood (as described below) while preventing theirdegradation into their pro-inflammatory fragments by extracellularendogenous protease. Methods of evaluating and discovering suitableanalogs or fragments of cathelicidin and other AMPs can, for example, bethrough the use of assays as disclosed in example 9 below.

Many different analogs of the above fragments can be made as for exampleas described in U.S. Pat. No. 4,242,256. There Compounds being analogsof a dipeptide in which the nitrogen atom of the linking amide group ofthe dipeptide is replaced by trivalent group and in which, optionally,the carbonyl function of this linking group is replaced by the divalentgroup—CH.sub.2—are of value in the synthesis of isosterically modifiedpeptides.

As used herein, the term “peptide” includes native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), such as peptoids and semipeptoids which are peptide analogs,which may have, for example, modifications rendering the peptides morestable while in a body or more capable of penetrating into target cells.Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992).

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as TIC, naphthylelanine (Nol),ring-methylated derivatives of Phe, halogenated derivatives of Phe oro-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

The peptides of the present invention can be utilized in a linear orcyclic form.

A peptide can be either synthesized in a cyclic form, or configured soas to assume a cyclic structure under suitable conditions.

For example, a peptide according to the teachings of the presentinvention can include at least two cysteine residues flanking the corepeptide sequence. In this case, cyclization can be generated viaformation of S—S bonds between the two Cys residues. Side-chain to sidechain cyclization can also be generated via formation of an interactionbond of the formula —(—CH2-)n-S—CH-2-C—, wherein n=1 or 2, which ispossible, for example, through incorporation of Cys or homoCys andreaction of its free SH group with, e.g., bromoacetylated Lys, Orn, Dabor Dap. Furthermore, cyclization can be obtained, for example, throughamide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn,di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at variouspositions in the chain (—CO—NH or —NH—CO bonds). Backbone to backbonecyclization can also be obtained through incorporation of modified aminoacids of the formulas H—N((CH2)n-COOH)—C(R)H—COOH orH—N((CH2)n-COOH)—C(R)H—NH2, wherein n=1-4, and further wherein R is anynatural or non-natural side chain of an amino acid.

Depending on the application and purpose, any of various AMPs/AMLs orcombinations of different AMPs/AML may be employed and/or regulated soas to practice the various embodiments of the present invention.Numerous examples of AMPs/AMLs suitable for use in the present inventionare listed on the Internet/World Wide Web athttp://www.bbcm.units.it/˜tossi/pag1.htm, and are described hereinbelow.

Examples of AMPs/AMLs include defensins, cathelicidins, andthrombocidins (alternately termed “platelet microbicidal proteins[PMPs]”).

Examples of defensins include alpha-defensins, beta-defensins, andneutrophil defensins.

Examples of alpha-defensins include alpha-defensin-1 to -6 (Mol Immunol.2003 November; 40(7):463-7; J Clin Invest. 1985 October; 76(4):1427-35).

Examples of beta-defensins include beta-defensin-1 (Genomics. 1997 Aug.1; 43(3):316-20; Biochem Biophys Res Commun 2002 Feb. 15; 291(1):17-22;FEBS Lett. 1995 Jul. 17; 368(2):331-5; Paulsen F et al., J Pathol. 2002November; 198(3):369-77), beta-defensin-2 (Biochemistry. 2001 Apr. 3;40(13):3810-6; Gene. 1998 Nov. 19; 222(2):237-44; Paulsen F et al., JPathol. 2002 November; 198(3):369-77), beta-defensin-3 (Cell Tissue Res.2001 November; 306(2):257-64; J Biol Chem. 2002 Mar. 8; 277(10):8279-89.Epub 2001 Dec. 11; J Biol Chem. 2001 Feb. 23; 276(8):5707-13; Paulsen Fet al., J Pathol. 2002 November; 198(3):369-77), beta-defensin-4 (JImmunol. 2002 Sep. 1; 169(5):2516-23), beta-defensin-5 (Am J Pathol.1998 May; 152(5):1247-58; J Biol Chem. 1992 Nov. 15; 267(32):23216-25),and beta-defensin-6 (FEBS Lett. 1993 Jan. 4; 315(2):187-92; Crit CareMed. 2002 February; 30(2):428-34).

Beta-defensins include those encoded by five conserved beta-defensingene clusters identified using a computational search strategy (SchutteB C. et al., 2002. Proc Natl Acad Sci USA. February 19; 99(4):2129-33).

Examples of neutrophil defensins include neutrophil alpha-defensins andneutrophil beta-defensins.

Examples of neutrophil alpha-defensins include neutrophilalpha-defensin-1//human neutrophil peptide (HNP)-1 (J Clin Invest. 1985October; 76(4):1436-9; Paulsen F et al., J Pathol. 2002 November;198(3):369-77), neutrophil alpha-defensin-2/HNP-2 (J Clin Invest. 1985October; 76(4):1436-9; Paulsen F et al., J Pathol. 2002 November;198(3):369-77), neutrophil alpha-defensin-3/HNP-3 (J Clin Invest. 1985October; 76(4):1436-9; Paulsen F et al., J Pathol. 2002 November;198(3):369-77), neutrophil alpha-defensin-4/HNP-4 (Mol Immunol. 2003November; 40(7):463-7), human defensin-5 (HD-5; D. E. Jones and C. L.Bevins, J. Biol. Chem. 267 (1992), pp. 23216-23225; J Biol Chem. 1992Nov. 15; 267(32):23216-25; Mol Immunol. 2003 November; 40(7):469-75;Quayle A J et al., Am. J. Pathol. 1998, 152:1247-1258; FEBS Lett. 1993Jan. 4; 315(2):187-92; D. E. Jones and C. L. Bevins, FEBS Lett. 315(1993); Paulsen F et al., J Pathol. 2002 November; 198(3):369-77), andhuman defensin-6 (HD-6; Mol Immunol. 2003 November; 40(7):463-7), humandefensin-5 (HD-5; D. E. Jones and C. L. Bevins, J. Biol. Chem. 267(1992), pp. 23216-23225; J Biol Chem. 1992 Nov. 15; 267(32):23216-25;Mol Immunol. 2003 November; 40(7):469-75; Quayle A J et al., Am. J.Pathol. 1998, 152:1247-1258; FEBS Lett. 1993 Jan. 4; 315(2):187-92; D.E. Jones and C. L. Bevins, FEBS Lett. 315 (1993); Paulsen F et al., JPathol. 2002 November; 198(3):369-77).

Examples of cathelicidins include LL-37/hCAP18 (LL-37) in humans (CurrDrug Targets Inflamm Allergy. 2003 September; 2(3):224-31; Eur JBiochem. 1996 Jun. 1; 238(2):325-32; Paulsen F et al., J Pathol. 2002November; 198(3):369-77). LL-37 is a 37 amino acid residue peptidecorresponding to amino acid residue coordinates 134-170 of its precursorhCAP18/human cathelicidin antimicrobial peptide protein (GenBank:ACCESSION NP_(—)004336; VERSION NP_(—)004336.2 GI:39753970; REFSEQ:accession NM_(—)004345.3). The proliferation and angiogenesis pathway ofLL-37 can be inhibited using pertussis toxin, an inhibitor of G-proteincoupled receptors (Koczulla, R. et al., 2003. J. Clin. Invest111:1665-1672). Similar AMPs/AMLs are listed in the following patentapplications: US 2003120037, US 200309626, US20020141620, US20020507, CA2383172, US 20020072495 and are incorporated by reference herein. Thehuman antibacterial cathelicidin precursor hCAP-18, is synthesized inmyelocytes and metamyelocytes and localizes to specific granules inneutrophils (Blood. 1997 Oct. 1; 90(7):2796-803).

Examples of AMP-like molecules include chemokines or fragments thereof.

Examples of such chemokines include CC chemokines and CXC chemokines.Considerable overlap of chemokine and AMP functions has beendemonstrated (Cole et al., 2001. J. Immunol. 167:623), and certainchemokines and defensins have actually been shown to bind to the samechemokine receptor, CCR6. Defensins and certain chemokines strikinglyshare similar characteristics, including size, disulfide bonding,interferon (IFN) inducibility, cationic charge, and more. Relevantsimilarities between chemokines and AMPs are described in the literature(refer, for example, to Durr and Peschel, 2002. Infection and Immunity70:6515). As such various chemokines and antibodies specific for suchchemokines may be employed in various applications of the presentinvention.

Examples of such CC chemokines include CCL1, CCL5/RANTES (Infect Immun2002 December; 70(12):6524-33; Eur J Biochem 1996 Apr. 1; 237(1):86-92),CCL8, CCL11, CCL17, CCL18, CCL19, CCL20/activation-regulated chemokine(LARC)/macrophage inflammatory protein-3alpha(MIP-3alpha)/Exodus-1/Scya20 (Yang D et al., Journal of LeukocyteBiology Volume 74, September 2003; 74(3):448-55), CCL21, CCL22, CCL25,CCL27/CTACK, and CCL28 (J Biol Chem. 2000 Jul. 21; 275(29):22313-23; JImmunol. 2003 Feb. 1; 170(3):1452-61). CCL chemokines are described inYang D et al., Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55.

Examples of such CXC chemokines include CXCL1, CXCL2, CXCL3, CXCL4(PF-4), CXCL7/NAP-2, CXCL8/IL-8, CXCL9 (MIG; Yang D et al., Journal ofLeukocyte Biology Volume 74, September 2003; 74(3):448-55), CXCL10/IP-10(The Journal of Immunology, 2001, 167: 623-627), CXCL11/IP-9/I-TAC (TheJournal of Immunology, 2001, 167: 623-627), CXCL12/SDF-1 (Yang D et al.,Journal of Leukocyte Biology Volume 74, September 2003; 74(3):448-55),CXCL13, CXCL14, connective tissue activating peptide 3 (CTAP-3; InfectImmun. 2002 December; 70(12):6524-33; Eur J Biochem 1996 Apr. 1;237(1):86-92), and CTAP-3 precursor platelet basic protein. CXCchemokines are described in Yang D et al., Journal of Leukocyte BiologyVolume 74, September 2003; 74(3):448-55.

Examples of fibrinopeptides include fibrinopeptide-A (Infect Immun. 2002December; 70(12):6524-33; Eur J Biochem 1996 Apr. 1; 237(1):86-92),fibrinopeptide-B (Infect Immun. 2002 December; 70(12):6524-33; Eur JBiochem 1996 Apr. 1; 237(1):86-92).

Examples of AMPs/AMLs further include XCL1 (Yang D et al., Journal ofLeukocyte Biology Volume 74, September 2003; 74(3):448-55), MIP-1beta(Yang D et al., Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55).

Further examples of AMPs/AMLs include adrenomedullin (Regul Pept. 2003Apr. 15; 112(1-3):147-52; J Biol Chem 1998 Jul. 3; 273(27):16730-8),alpha-melanocyte stimulating hormone (Cutuli M et al., J Leukoc Biol.2000 February; 67(2):233-9; Neuroimmunomodulation-2002-2003;10(4):208-16), an angiogenin (Nature Immunology, March 2003),angiogenin-4 (Nature Immunology, March 2003), antibacterial peptidesB/enkelytin (Neuroimmunol 2000 Sep. 22; 109(2):228-35),antileukoprotease (ALP; Biochem Biophys Res Commun 1998 Jul. 30;248(3):904-9; Am J Respir Crit Care Med 1999 July; 160(1):283-90), alymphokine-activated killer cell-derived antimicrobial peptide, aplatelet-derived antimicrobial peptide, antimicrobial peptide PR39, anapolipoprotein, an apolipoprotein-C, apolipoprotein-C2 (HypertensPregnancy 2002; 21(3):199-204; Peptides. 2000 March; 21(3):327-30),apolipoprotein-C3 (Hypertens Pregnancy 2002; 21(3):199-204; Peptides.2000 March; 21(3):327-30), an apolipoprotein-E (Hypertens Pregnancy2002; 21(3):199-204; Peptides. 2000 March; 21(3):327-30),apolipoprotein-E2 (Brain Res 1997 Feb. 21; 749(1):135-8; Biochemistry2002 Oct. 1; 41(39):11820-3; Eur J Clin Chem Clin Biochem 1997 August;35(8):581-9), a bactericidal/permeability-increasing protein (Paulsen Fet al., J Pathol. 2002 November; 198(3):369-77; Mol Microbiol 1995 Aug.17:523-31; J Biol Chem 1987 Nov. 5; 262(31):14891-4), a bonemorphogenetic protein (BMP), BMP-2/4, BMP-5, buforin, calcitermin (FEBSLett. 2001 Aug. 24; 504(1-2):5-10), a cathepsin, cathepsin B, cathepsinG, cathepsin K, a lysosomal cathepsin, a chromogranin (Blood 2002 Jul.15; 100(2):553-9), chromogranin A (Blood 2002 Jul. 15; 100(2):553-9),chromogranin B (Blood 2002 Jul. 15; 100(2):553-9), chymase (Immunology2002 April; 105(4):375-90), connective tissue activating peptide-3,cystatin (APMIS. 2003 November; 111(11):1004-1010; Biol Chem HoppeSeyler 1988 May; 369 Supp1:191-7), DCD-1 (J Immunol Methods. 2002 Dec.1; 270(1):53-62), dermicidin (Nat Immunol. 2001 December; 2(12):1133-7),elastase-specific inhibitor/SKALP (skin-derivedantileucoproteinase)/elafin (Biochem Soc Trans. 2002 April; 30(2):111-5;J Invest Dermatol 2002 July; 119(1):50-5), eNAP-1, eosinophil cationicprotein (Peptides. 2003 April; 24(4):523-30; J Immunol 2002 March168:2356-64; Eur J Biochem 1996 Apr. 1; 237(1):86-92; Peptides. 2003April; 24(4):523-30; J Exp Med 1989 Jul. 1; 170(1):163-76), ESC42,ESkine, FALL-39 (Proc Natl Acad Sci USA. 1995 Jan. 3; 92(1):195-9), Fasligand (FasL; Berthou C et al., J Immunol. 1997 Dec. 1;159(11):5293-300), fractalkine, a glycosaminoglycan, granulysin (ReprodBiol Endocrinol. 2003 Nov. 28; J Immunol. 2003 Mar. 15; 170(6):3154-61;Cancer Immunol Immunother. 2002 January; 50(11):604-14. Epub 2001November; Expert Opin Investig Drugs. 2001 February; 10(2):321-9),granzyme B (Berthou C et al., J Immunol. 1997 Dec. 1; 159(11):5293-300),HAX-1, heparin binding protein/CAP37 (Paulsen F et al., J Pathol. 2002November; 198(3):369-77; J Clin Invest 1990 May; 85(5):1468-76), ahepcidin (J Biol Chem. 2001 Mar. 16; 276(11):7806-10. Epub 2000 Dec. 11;Eur J Biochem 2002 April 269:2232-7), an HE2, HE2alpha (Biol Reprod.2002 September; 67(3):804-13), an HE2alpha C-terminal fragment (BiolReprod. 2002 September; 67(3):804-13), HE2beta1 (Biol Reprod. 2002September; 67(3):804-13), an HE2-gene derived transcript, histatin(Antimicrob Agents Chemother 2001 December 45:3437-44; Biochem CellBiol. 1998; 76(2-3):247-56), a histone, histone H2A, histone H-2b(Peptides. 2003 April; 24(4):523-30; J Immunol 2002 March 168:2356-64;Eur J Biochem 1996 Apr. 1; 237(1):86-92), HMG-17, HtpG, an HtpG homolog,HS1 binding protein, interleukin-8, lactoferrin (Eur J Nucl Med. 2000March; 27(3):292-301; Paulsen F et al., J Pathol. 2002 November;198(3):369-77; J Mammary Gland Biol Neoplasia 1996 July; 1(3):285-95), alymphokine-activated killer (LAK) cell AMP (Hua Xi Yi Ke Da Xue Xue Bao2002 January; 33(1):87-90), lysozyme (Paulsen F et al., J Pathol. 2002November; 198(3):369-77; Anat Embryol (Berl) 2002 July; 205(4):315-23),a macrophage inflammatory protein (MIP), MIP-1alpha, MIP-1beta,MIP-3alpha, a mast cell granule serine proteinase (Immunology 2002April; 105(4):375-90), a matrix metalloproteinase (MMP), MMP-2, MMP-7(Paulsen F et al., J Pathol. 2002 November; 198(3):369-77), migrationinhibitory factor (J Immunol. 1998 Sep. 1; 161(5):2383-90; Scand JInfect Dis. 2003; 35(9):573-6), MMP-9, MRP8 (Behring Inst Mitt. 1992April; (91):126-37), MRP14 (Behring Inst Mitt. 1992 April; (91):126-37),neutrophil gelatinase-associated lipocalin (NGAL; Exp Dermatol. 2002December; 11(6):584-91; Mol Cell. 2002 November; 10(5):1033-43),neutrophil lysozyme (Int J Antimicrob Agents. 1999 September;13(1):47-51), an opioid peptide, perforin (Berthou C et al., J Immunol.1997 Dec. 1; 159(11):5293-300), phospholipase A(2) (PLA(2); Peptides.2003 April; 24(4):523-30; J Exp Med 1989 Jul. 1; 170(1):163-76),platelet basic protein (Infect Immun 2002 December; 70(12):6524-33; EurJ Biochem 1996 Apr. 1; 237(1):86-92), platelet factor-4, psoriasin (JHistochem Cytochem. 2003 May; 51(5):675-85; Glaser R et al., J InvestDermatol 117: 768(abstr 015)), retrocyclin (Proc Natl Acad Sci USA 2002Feb. 19; 99(4):1813-8), secretory leukocyte proteinase inhibitor (SLPI;Shugars D C et al., Gerontology. 2001 September-October; 47(5):246-53;Biochem Soc Trans. 2002 April; 30(2):111-5; J Invest Dermatol 2002 July;119(1):50-5), secretory phospholipase A(2) (Peptides. 2003 April;24(4):523-30; J Immunol 2002 March 168:2356-64; Eur J Biochem 1996 Apr.1; 237(1):86-92; Paulsen F et al., J Pathol. 2002 November;198(3):369-77), substance P, an S100 calcium-binding protein, S100A7,S100A8, S100A9, a thymosin, thymosin beta-4 (Infect Immun. 2002December; 70(12):6524-33; Eur J Biochem 1996 Apr. 1; 237(1):86-92;Infect Immun. 2002 December; 70(12):6524-33; Eur J Biochem 1996 Apr. 1;237(1):86-92), thymus and activation-regulated chemokine (TARC), TL1A,tryptase (Immunology 2002 April; 105(4):375-90), ubiquicidin (Eur J NuclMed. 2000 March; 27(3):292-301; Hiemstra P S, van den Barselaar M T etal., J Leukocyte Biol 1999; 66: 423-428; J Nucl Med 2001 May 42:788-94),and urokinase-type plasminogen activator.

The AMP/AML may any one of 28 potential candidates for defensin likepeptides which were computationally discovered. (Am J Respir Cell MolBiol. 2003 July; 29(1):71-80).

As described hereinabove, the present invention can be used to treat anyof various diseases which are associated with: (i) a tumor; (ii)inflammation; (iii) a wound; (iv) autoimmunity; (v) dysregulation ofgrowth/differentiation of a cell/tissue; (vi) dysregulation ofgrowth/differentiation balance of a cell/tissue; and/or (vii)angiogenesis.

Examples of diseases which can be treated according to the presentinvention are listed in International Pub. No. WO 2004-056307.

Examples of diseases which can be treated according to the presentinvention are also as follows.

Examples of tumors include a skin tumor, Osteosarcoma, Ewing's sarcoma,Chondrosarcoma, Malignant fibrous histiocytoma, Fibrosarcoma, Chordoma,osteoid osteoma, osteoblastoma, osteochondroma, enchondroma,chondromyxoid fibroma, and giant cell tumor, lymphoma and multiplemyeloma, a keratinocytic tumor, a gastrointestinal tumor, a carcinoma, amelanoma, a squamous cell tumor, oral squamous cell carcinoma, lymphoma,a malignant tumor, a benign tumor, a solid tumor, a metastatic tumor anda non-solid tumor.

The concentration of human beta-defensin-2 in oral squamous cellcarcinoma is much higher than in normal oral epithelium (Sawaki, K. etal., 2002. Anticancer Res. 22:2103-2107). There is a genetic linkbetween proliferation of cells and cancer Impairment of regulation ofproliferation and differentiation lead to cancer development. Adeveloping tumor needs help from neighboring cells in order to becomecancerous. Overexpression or overactivity of cytokines is involved inorchestrating these processes. Continuous assault by chronicinflammation contributes to the transformation of cells as well.Angiogenesis is an important process for cancer development. AMPs areinductors of angiogenesis (Koczulla, R. et al., 2003. J. Clin. Invest111:1665-1672). Therefore inhibiting differentiation and proliferationas well as angiogenesis by antagonists to AMPs and cytokines can be usedto treat cancer. Urokinase-type plasminogen activator (uPA), hasantimicrobial properties (Gyetko, M R. et al., 2002. J. Immunol.168:801-809) and is involved in metastatic spreading of malignant cells.The in vitro and in vivo findings suggest that alpha-defensins arefrequent peptide constituents of malignant epithelial cells in renalcell carcinoma with a possible direct influence on tumor proliferation(Muller, C A. et al., 2002. Am. J. Pathol. 160:1311-1324). Certainanti-angiogenic compounds were found to have potent anticancer propertyin vivo experimental studies. Therefore inhibition of angiogenic AMPssuch as LL-37 is one form of treatment for cancer. Matrixmetalloproteinases (MMPs) are known to play an important role inextracellular matrix remodeling during the process of tumor invasion andmetastasis. Overexpression of MMP-2 and MMP-9 proteins was observed in alarge percentage of ESCC tumors, respectively localized in tumor cellcytoplasm and stromal elements (J Cancer Res Clin Oncol. 2003 Oct. 16).

BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis oforal carcinoma cells (Overexpression of BMP-2/4, -5 and BMPR-IAassociated with malignancy of oral epithelium Oral Oncol. 2001,37:225-33.)

Examples of diseases include an idiopathic/inflammatory disease, achronic/inflammatory disease, an acute/inflammatory disease, aninflammatory cutaneous disease, an inflammatory gastrointestinaldisease, a tumor associated with inflammation, an allergic disease, anautoimmune disease, an infectious disease, a malignant disease, atransplantation related disease, an inflammatory degenerative disease,an injury associated with inflammation, a disease associated with ahypersensitivity, an inflammatory cardiovascular disease, aninflammatory glandular disease, an inflammatory hepatic disease, aninflammatory neurological disease, an inflammatory musculo-skeletaldisease, an inflammatory renal disease, an inflammatory reproductivedisease, an inflammatory systemic disease, an inflammatory connectivetissue disease, an inflammatory neurodegenerative disease, necrosis, aninflammatory disease associated with an implant, an inflammatoryhematological disease, an inflammatory eye disease, an inflammatoryrespiratory disease.

Examples of cutaneous/inflammatory diseases include psoriasis, dandruff,pemphigus vulgaris, lichen planus, atopic dermatitis, excema,scleroderma, dermatomyositis, alopecia, blepharitis, skin carcinoma,melanoma, squamous cell carcinoma, acne vulgaris, erythema toxicumneonatorum, folliculitis, skin wrinkles, autoimmune bullous skindisease, bullous pemphigoid, pemphigus foliaceus, dermatitis, and drugeruption.

Examples of gastrointestinal/inflammatory diseases include Crohn'sdisease, chronic autoimmune gastritis, autoimmune atrophic gastritis,primary sclerosing cholangitis, autoimmune achlorhydra, colitis,ileitis, chronic inflammatory intestinal disease, inflammatory bowelsyndrome, chronic inflammatory bowel disease, celiac disease, an eatingdisorder, gallstones and a gastrointestinal ulcer.

Crohn's disease is an inflammatory bowel disease. Since the bowel isexposed to the outer environment, the importance of AMPs as part of itsdefense and normal cellular regulation is important, as in skin, and theactivity of the AMPs plays an important role in the normal physiology aswell as pathological conditions in these tissues. Abnormalities in theexpression and/or activity of the AMPs will contribute to pathologies inthese tissues. Paneth cells (a specific type of cell in the intestine)are required to help promote normal vessel formation in cooperation withbacteria—mice absent Paneth cells were incapable of appropriate bloodvessel formation. Of note, colonization by one particular type ofbacteria commonly found in normal mouse and human intestine, calledBacteroides thetaiotaomicron, or B. thetaiotaomicron, stimulated bloodvessel development as efficiently as implantation of a whole microbialsociety. The conclusion, B. thetaiotaomicron and Paneth cells worktogether to stimulate postnatal blood vessel formation. The ability ofAMPs to act as chemoattractants for cells of the innate- andadaptive-immune system plays an important role in perpetuating chronicinflammation in the gastrointestinal tract (Cunliffe, R N, Mahida, Y R.,2003. J Leukoc Biol. October 2 [Epub ahead of print]). The AMP LL-37,beta-defensins, human alpha-defensins, beta-defensins (including HD5),HN-6, lysozyme and secretory PLA2, TL1A, are expressed in Paneth cellsand intestine, secretory epithelial cells in the small intestine (Ghosh,D. et al., 2002. Nat. Immunol. 3:583-590; Fellermann, K. et al., 2003.Eur. J. Gastroenterol. Hepatol. 15:627-634). Where alpha-defensins areoverexpressed, they are chemoattract naive T and immature dendriticcells and dendritic cells and monocytes (Yang, D. et al., 2000. J.Leukoc. Biol. 68:9-14; Risso, A., 2000. J. Leukoc. Biol. 68:785-792;Territo, M C. et al., 1989. J. Clin. Invest 84:2017-2020). Humanalpha-defensins as well as other AMPs contribute to local intestinalhost defense as part of innate immunity and may be of major relevance inmicrobial infection and chronic inflammatory bowel disease (Wehkamp, J.et al., 2002. Dig. Dis. Sci. 47:1349-1355). The alpha-defensins convertan acute inflammation to a chronic inflammation by downregulating humanpolymorphonuclear leukocyte chemotaxis, for example,alpha-defensin-1/human neutrophil protein-1, acts as an antichemotacticagent for human polymorphonuclear leukocytes). It is known that chronicinflammation is commonly characterized by the presence of increased cellproliferation and connective tissue than exudate with the presence oflymphocytes and plasma cells rather than polymorphonuclear leukocytes.Thus, suitable regulation of such AMPs/AMLs can be used to treatdiseases such as inflammatory bowel disease, Crohn's disease andulcerative colitis.

Gastritis is an inflammatory condition of the stomach. There are twomain forms of gastritis, A and B. Gastritis type A is considered todevelop in an autoimmune process. In both types there is a role forinfectious agents such as Helicobacter pylori. AMPs are involved in bothprocesses. Defensins are involved in pathogenesis of gastritis(Bajaj-Elliott, M. et al., 2002. Gut 51:356-361). Thus, suitableregulation of such AMPs/AMLs can be used to treat diseases such asgastritis.

Examples of allergic/inflammatory diseases include asthma, hives,urticaria, a pollen allergy, a dust mite allergy, a venom allergy, acosmetics allergy, a latex allergy, a chemical allergy, a drug allergy,an insect bite allergy, an animal dander allergy, a stinging plantallergy, a poison ivy allergy, anaphylactic shock, anaphylaxis, atopicallergy and a food allergy.

Examples of hypersensitivity include Type I hypersensitivity, Type IIhypersensitivity, Type III hypersensitivity, Type IV hypersensitivity,immediate hypersensitivity, antibody mediated hypersensitivity, immunecomplex mediated hypersensitivity, T lymphocyte mediatedhypersensitivity, delayed type hypersensitivity, helper T lymphocytemediated hypersensitivity, cytotoxic T lymphocyte mediatedhypersensitivity, TH1 lymphocyte mediated hypersensitivity, and TH2lymphocyte mediated hypersensitivity.

Examples of cardiovascular/inflammatory and/or inflammatoryhematological diseases include atherosclerosis, Takayasu's arteritis,polyarteritis nodosa, Raynaud's phenomenon, temporal arteritis,inflammatory anemia, inflammatory lymphopenia, pernicious anemia,occlusive disease, myocardial infarction, thrombosis, Wegener'sgranulomatosis, lymphoma, leukemia, Kawasaki syndrome, anti-factor VIIIautoimmune disease, necrotizing small vessel vasculitis, microscopicpolyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizingglomerulonephritis, crescentic glomerulonephritis, antiphospholipidsyndrome, antibody induced heart failure, thrombocytopenic purpura,autoimmune hemolytic anemia, cardiac autoimmunity, Chagas' disease,iron-deficiency anemia, and anti-helper T lymphocyte autoimmunity.

Inflammation is part of the pathological process leading to thedevelopment of atherosclerosis. Chlamydia pneumonia as well as othervarious microorganisms serve as potential etiological factors, linkinginflammation and atherosclerosis. Inflammation is a predisposing factoras well as a consequence of several CNS pathologies. Inflammation ispart of the pathophysiologic processes occurring after the onset ofcerebral ischemia in ischemic stroke, as well as other CNS pathologiessuch as head injury and subarachnoid hemorrhage. In addition,inflammation in the CNS or in the periphery by itself is considered as arisk factor for the triggering the development of cerebral ischemia.Endothelial cells express and secrete AMPs. Cationic antimicrobialprotein of 37 kDa (CAP37) also termed heparin binding protein,originally isolated from human neutrophils, is an importantmultifunctional inflammatory mediator is expressed within the vascularendothelium associated with atherosclerotic plaques (Lee, T D. et al.,2002. Am. J. Pathol. 160:841-848). Human beta-defensin-2 is expressed byastrocytes and its expression is increased in response to cytokines andLPS (Hao, H N. et al., 2001. J. Neurochem. 77:1027-1035). Therefore, AMPregulation can be used for treatment or prevention of these conditions.

Anemia associated with inflammatory chronic diseases is one of thebody's methods of fighting pathogens by reducing available intercellular iron uptake of pathogens. Iron is absorbed by neutrophils.Sometimes chronic inflammation can occur without the presence ofpathogens. Under chronic inflammatory conditions, cytokines induce adiversion of iron traffic leading to hypoferremia. Such as in chronicbacterial endocarditis, osteomyelitis, juvenile rheumatoid arthritis,rheumatic fever, Crohn's disease, and ulcerative colitis and Chronicrenal failure. Transferrin bound iron transports to monocytes causinganemia. This “transport” is thought to be related to AMP activity.Cytokines IL-1, IL-6 and TNF-beta initiate defensin production anddefensin initiate the cytokine production, the result being iron overabsorption by monocytes. The regulation of iron transport by cytokinesis a key mechanism in the pathogenesis of anemia of chronic disease(Ludwiczek, S. et al., 2003. Blood 101:4148-4154). Therefore, regulationof AMPs can be used to regulate iron level homeostasis. Hepcidin AMP isknown to regulate iron uptake, therefore inhibiting hepcidin can be usedto increase iron absorption (Nicolas, G. et al., 2002. Blood Cells Mol.Dis. 29:327-335). However, there are other AMPs indirectly involved iniron regulation such as defensin and LL-37. Since HNP-1 is anon-specific defensive peptide present in neutrophils, it plays animportant role in the protection against diseases such as oral lichenplanus, leukoplakia, and glossitis associated with iron deficiency(Mizukawa, N. et al., 1999. Oral Dis. 5:139-142). Likewise all cationicneutrophil derived AMPs would induce iron hypoferremia when overexpressed. Therefore regulating of these AMPs can be used to treat suchdiseases.

Leukocyte SLPI (secretory leukocyte proteinase inhibitor (SLPI))expression seems to be up-regulated in active Wegner's granulomatosis,therefore inhibiting its activity can be used to treat diseases such asWegener's granulomatosis and other types of vasculitis

Examples of glandular/inflammatory diseases include type I diabetes,type II diabetes, type B insulin resistance, Schmidt's syndrome,Cushing's syndrome, thyrotoxicosis, benign prostatic hyperplasia,pancreatic disease, Hashimoto's thyroiditis, idiopathic adrenal atrophy,Graves' disease, androgenic alopecia, thyroid disease, thyroiditis,spontaneous autoimmune thyroiditis, idiopathic myxedema, ovarianautoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis,Addison's disease, and Type I autoimmune polyglandular syndrome.

Diabetes mellitus is a systemic disease with several major complicationsaffecting both the quality and length of life. One of thesecomplications is periodontal disease (periodontitis). Periodontitis ismuch more than a localized oral infection. (Iacopino, A M., 2001. Ann.Periodontol. 6:125-137). When diabetes mellitus is under therapeuticcontrol, periapical and other lesions heal as readily as in nondiabetics(Bender, I B, Bender, A B. et al., 2003. J. Endod. 29:383-389). Recentstudies on diseases which involve insulin insensitivity (e.g. obesity,type 2 diabetes and atherosclerosis) also show increased cytokineproduction and markers of inflammation. Evidence at present favorschronic inflammation as a trigger for chronic insulin insensitivity,rather than the reverse situation. (Grimble, R F., 2002. Curr. Opin.Clin. Nutr. Metab Care 5:551-559). Recent human studies have establisheda relationship between high serum lipid levels and periodontitis.Possible causes are a high glucose levels (such as hyperglycemia ofdiabetics) with added LDL levels such as in high diabetic patients areprone to elevated low density lipoprotein cholesterol and triglycerides(LDL/TRG) even when blood glucose levels are well controlled, lead toLPS-like bondings that induce AMP overexpression. Thus, the presentinvention can be used to treat diabetes and diabetes related diseasessuch as periodontitis and diabetes associated healing deficiencies.

Proliferative retinopathy is one of the chronic complications ofdiabetes. The process includes the development of abnormal blood vesselsthat might lead to retinal detachment and blindness. LL37 and other AMPsare involved in angiogenesis (Koczulla, R. et al., 2003. J. Clin. Invest111:1665-1672), therefore LL-37 regulation can be used to prevent thedevelopment of newly formed blood vessels and therefore for preventingdiabetes related eye diseases.

Examples of hepatic inflammatory diseases include primary biliarycirrhosis, active chronic hepatitis, lupoid hepatitis, autoimmunehepatitis, and hepatic cirrhosis.

Examples of neurological inflammatory diseases include neurodegenerativedisease, multiple sclerosis, Alzheimer's disease, Parkinson's disease,myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmuneneuropathy, Lambert-Eaton myasthenic syndrome, paraneoplasticneurological disease, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy,Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmuneneuropathy, acquired neuromyotonia, arthrogryposis multiplex, opticneuritis, spongiform encephalopathy, migraine, headache, clusterheadache, and stiff-man syndrome.

With respect to multiple sclerosis (MS), defensins and lactoferrinsexist in cerebrospinal fluid (CSF). These peptides have antimicrobialexpression in some diseases like pneumonia and meningitis, which maytrigger a pathway. It seems that pathways to MS are similar torheumatoid arthritis where AMPs reside in the synovial fluid surroundingthe joint. Peptides involved are amongst others: IP-10, defensins andlactoferrins, CAP37.

Examples of connective tissue inflammatory diseases include arthritis,rheumatoid arthritis, pyogenic arthritis, mixed connective tissuedisease, cholesteatoma, relapsing polychondritis, autoimmune myositis,primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis,tendinitis, a ligament inflammation, chondritis, a joint inflammation, asynovial inflammation, carpal tunnel syndrome, osteoarthritis,ankylosing spondylitis, a skeletal inflammation, an autoimmune eardisease, osteoporosis, fibromyalgia, periodontitis, and an autoimmunedisease of the inner ear.

With respect to diseases such as arthritis, AMPs are expressed andproduced in healthy and inflamed human synovial membranes. Deposition ofthe AMPs lysozyme, lactoferrin, secretory phospholipase A(2) (sPA(2)),matrilysin (MMP7), human neutrophil alpha-defensin-1, -2, and -3, humanbeta-defensin-1, and human beta-defensin-2 was determined byimmunohistochemistry. Expression of mRNA for the AMPs bactericidalpermeability-increasing protein (BPI), heparin binding protein, LL37,human alpha-defensin-5, human alpha-defensin-6, and humanbeta-defensin-1, -2, and -3 was analyzed by reversetranscription-polymerase chain reaction (RT-PCR). RT-PCR revealed CAP37and human beta-defensin-1 mRNA in samples of healthy synovial membrane.Additionally, human beta-defensin-3 and/or LL37 mRNA was detected insynovial membrane samples from patients with pyogenic arthritis (PA),osteoarthritis (OA) or rheumatoid arthritis (RA). Immunohistochemistryhas identified lysozyme, lactoferrin, sPA(2), and MMP7 in type Asynoviocytes of all samples. Human beta-defensin-1 was only present intype B synoviocytes of some of the samples Immunoreactive humanbeta-defensin-2 peptide was only visible in some inflamed samples.HNP1-3 was detected in both healthy and inflamed synovial membranes. Thedata suggest that human synovial membranes produce a broad spectrum ofAMPs. Under inflammatory conditions, the expression pattern changes,with induction of human beta-defensin-3 in PA (LL37 in RA; humanbeta-defensin-3 and LL37 in OA) as well as down-regulation of humanbeta-defensin-1 (Paulsen, F. et al., 2002. J. Pathol. 198:369-377;Cunliffe, R N, Mahida, Y R., 2003. J Leukoc Biol. October 2 [Epub aheadof print]). Thus regulating one or more of these AMPs or their activitywill inhibit the pathological process in a disease such as arthritis.

Microbial mixed keratin-biofilms in cholesteatomas are caused by AMPswhich are overexpressed (Jung, H H. et al., 2003. Laryngoscope113:432-435; Chole, R A, Faddis, B T., 2002 Arch. Otolaryngol. Head NeckSurg. 128:1129-1133), AMPs such as LL-37 or other defensins or otherAMPs are involved. Therefore, suitable regulation of such AMPs can beused for treating diseases such as cholesteatomas.

Examples of inflammatory renal diseases include diabetic nephropathy.

Examples of inflammatory reproductive diseases include repeated fetalloss, ovarian cyst, or a menstruation associated disease.

Examples of inflammatory systemic diseases include systemic lupuserythematosus, systemic sclerosis, septic shock, toxic shock syndrome,Reiter's syndrome, and cachexia.

Examples of inflammatory infectious diseases include candidiasis, afungal infection, mycosis fungoides, a chronic infectious disease, asubacute infectious disease, an acute infectious disease, a viraldisease, a bacterial disease, a protozoan disease, a parasitic disease,a mycoplasma disease, gangrene, sepsis, a prion disease, influenza,tuberculosis, bacterial pneumonia, malaria, acquired immunodeficiencysyndrome, chronic fatigue syndrome, and severe acute respiratorysyndrome.

Examples of transplantation related/inflammatory diseases include graftrejection, chronic graft rejection, subacute graft rejection, acutegraft rejection hyperacute graft rejection, rejection of an implant andgraft versus host disease.

Examples of implants include a prosthetic implant, a breast implant, asilicone implant, a dental implant, a penile implant, a cardiac implant,an artificial joint, a bone fracture repair device, a bone replacementimplant, a drug delivery implant, a catheter, a pacemaker, an artificialheart, an artificial heart valve, a drug release implant, an electrode,and a respirator tube.

Examples of injuries associated with inflammation include a skin wound,an abrasion, a bruise, a cut, a puncture wound, a laceration, an impactwound, a concussion, a contusion, a thermal burn, frostbite, a chemicalburn, a sunburn, a desiccation, a radiation burn, a radioactivity burn,a smoke inhalation, a torn muscle, a pulled muscle, a torn tendon, apulled tendon, a pulled ligament, a torn ligament, a hyperextension, atorn cartilage, a bone fracture, a pinched nerve and a gunshot wound.

Examples of inflammatory respiratory diseases include asthma, allergicasthma, diffuse panbronchiolitis, emphysema, idiopathic pulmonaryfibrosis, cystic fibrosis, influenza, sinusitis, sinusitis and chronicobstructive pulmonary disease.

Examples of inflammatory eye diseases include dry-eye disease,phacogenic uveitis, blepharitis and sympathetic ophthalmia.

Dry eye disease is a chronic inflammatory eye disease. Is particularlyan issue for post-menopausal women, the elderly, and patients withsystemic diseases such as Sjogren's syndrome, rheumatoid arthritis,lupus and diabetes (37% of people with diabetes suffer from the diseaseand 28% of adults having the disease). Defensins act as chemokines toT-cells (Stern, M E, et al., 2002. Invest Ophthalmol. Vis. Sci.43:2609-2614).

For identifying and classifying disease, a kit comprising a reagentuseful for identifying the level of cathelicidin in blood foridentifying diseases types is included. The kit is compartmentalized toreceive one or more of (i) an oligonucleotide for detection of acathelicidin or fragment thereof; or (ii) an antibody for detection ofcathelicidin or a fragment thereof.

As described above, preventing binding of AMPs/AMLs to cognate receptorsby using ananlogues of same AMPs that compete with binding to samereceptors without inducing the disease may be used to inhibit abiological process mediated by binding of the AMP/AML to the receptor.Over 50 AMPs/AMLs and over 20 receptors thereof are involved diseasepathogenesis, therefore inhibiting correct target combinations of ligandand receptors is essential for treatment of such diseases. Examples ofsuch AMPs/AMLs and cognate receptors thereof, and examples of the typesof diseases which can be treated using this approach are shown in Table1.

Ample guidance for practicing methods and techniques of the presentinvention, and for obtaining and utilizing materials employed forpracticing the present invention is provided in the literature of theart (refer, for example, to U.S. Patent Application No. 20030044907).

Thus, the present invention enables for the first time relative to theprior art, treatment of any of various diseases by AMPs in particular bycathelicidin. The present invention clearly shows how cathelicidin isassociated with biological processes in cells/tissues such asdysregulated growth/differentiation, dysregulated growth/differentiationbalance, inflammation, and angiogenesis and autoimmunity. UsingAMPs/AMLs, and/or inhibitors of pro-inflammatory fragments thereof isneeded for treatment of disease.

Further, in addition to treatment with AMPs, such as cathelicidin, andfunctional fragments and analogs thereof, the invention also provides anew medical use for the treatment of obesity and/or excess body weightthat includes the administration of a therapeutically effective amountof one or more LPS neutralizing compounds selected from the groupconsisting of: BPI (bactericidal/permeability-increasing protein) andfragments and variations thereof such as Neuprex™ (rBPI21, opebacan) amodified recombinant fragment of BPI and Mycoprex™ (both XomaCorporation); protegrins such as protegrin-1; lactoferrins such aslactoferricin; Nisin(s) and their variants (Mol Microbiol. 2008 July;69(1):218-30); Heliomicin and its variants (e.g., ETD151) (InternationalJournal of Antimicrobial Agents 25 (2005) 448-452; Biochemistry. 2001Oct. 9; 40(40):11995-2003); magainin (Biochemistry. 2003 Oct. 28;42(42):12251-9); Colistin (polymyxin E), Polymyxin b(polymyxin bsulfate) and polymyxin derivatives (Antimicrobial Agents Chemother. 2008Jun. 30); Antiendotoxin antibody; Curcumin and lipopolysaccharidebinding peptides; Lipid A analogs; phospholipid emulsion; and ethylpyruvate (Curr Opin Anaesthesiol. 2008 April; 21(2):98-104). Therefore,with regards to treating obesity, diabetes and overweight, the term“cathelicidin” and the use thereof will include any of the above LPSneutralizing antimicrobials.

It is expected that during the life of this patent many relevant drugscreening techniques will be developed and the scope of the phrase“method of identifying a compound” is intended to include all such newtechnologies a priori.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

In the examples that follow, all animal work was performed underguidelines approved by either the Hebrew University of Jerusalem AnimalCare and Ethics Committee (EAE model, obesity model, insulin resistanceand periodontitis model) and the University of Tennessee USA (CIA modeland osteoporosis model).

Example 1

Use of cathelicidins for optimal treatment of arthritis and rheumaticdiseases such as rheumatoid arthritis which are associated withinflammation, autoimmunity.

Background:

Diseases associated with inflammation, autoimmunity and/or skincell/tissue proliferation/differentiation imbalance include numerousdiseases, such as arthritis, for which no optimal therapy exists.Cathelicidin hCAP-18 pro-sequence and its active form (LL-37) isexpressed in the bone marrow.

The present inventors have hypothesized that regulating such AMPs/AMLsas cathelicidin may be used for treating diseases such as arthritis.While reducing the present invention to practice, a method of using the34a.a. cathelicidin (mCRAMP) (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) (SEQNO 61) for optimal treatment in an Collagen induced arthritis mousemodel of the human disease associated with inflammation, autoimmunitysuch as in rheumatoid arthritis, Sjogren's, scleroderma,dermatomyositis, Systemic Lupus Erythematosus, sarcoidosis wasdemonstrated for the first time, as described below, thereby overcomingthe limitations of the prior art.

The present in-vivo experiment uses a mouse model of collagen inducedarthritis.

Materials:

Antimicrobial peptides (AMPs): Synthetic peptides for the MouseCathelicidin mCRAMP 34a.a. was purchased from Biosight Ltd (Israel).Chick native CII purchase from Sigma or Chondrex), a 1(II) chains orCB11 fragment of CII. 10 mM acetic acid, filter sterilized with 0.2-umfilter Incomplete Freund's adjuvant (IFA; e.g., Difco). Mycobacteriumtuberculosis (strain H37Ra; heat-killed; available by writing toMinistry of Agriculture, Fisheries, and Food, Central VeterinaryLaboratory, Weybridge, Surrey, United Kingdom). DBA/1JLacJ mice (JacksonLabs)

Methods:

The Protocol for this model is described in the publication NatureProtocols (Brand D D et al. 2007; 2(5):1269-75).

Briefly, Male DBA/1 mice, 9-11 weeks of age were used in theseexperiments. Mice were divided into two groups, experimental andcontrol, and each mouse was immunized at the base of the tail with 50 μlcontaining 100 μg of bovine CII emulsified in complete Freund'ssupplemented to 4 mg/ml of heat killed mycobacterium. Mice wereanesthetized during the immunization by inhaled isofluorane. On the sameday as the immunization, mice also received their first injection of thevehicle (150 mM saline, control group) or experimental peptide(experimental group) at a concentration of 1.5 mg/kg. Subsequently ondays 2 and 4 post immunization, the dose was reduced to 1.0 mg/kg.Starting with day 7 and through day 72, a dose of 0.8 mg/kg was used.All treatments were performed 3 times per week, on a Monday, Wednesday,and Friday schedule, and the peptide or control vehicle was administeredintraperitoneally for each treatment, rotating injection areas. All micewere weighed at the beginning of the experiment in order to calculatedosage administered. Mice were weighed on days 0, 21 and 46. At day4946, the mice were again weighed (average of 1.6 gm increase andcontrol group had a significantly greater increase in weight than didtreatment group) and dosages were adjusted accordingly.

Starting on day 11, all mice were examined 3 times per week forincidence and severity of arthritis. and each arthritic limb wasassigned a numerical score based on the degree of inflammation observedaccording to the scale below.

Severity scoring system is as described in the publication (Rosleniec Eet al. Current protocols in immunology, 1997). Briefly, Score 0 Noevidence of erythema or swelling. Score 1 Erythema and mild swellingconfined to the tarsals or ankle joint. Score 2 Erythema and mildswelling extended from the ankle to the tarsals. Score 3 Erythema andmoderate swelling extended from the ankle to the metatarsal joints.Score 4 Erythema and severe swelling encompass the ankle, foot anddigits or ankylosis of the joint. In this experiment, two groups of mice(10 in treatment and 11 in control) were compared. The control groupreceived saline in parallel with treatment group on same days as thetreatment group received mCRAMP (0.8 mg/Kg) intraperitoneuly 3 times aweek.

Experimental Results and Statistical Analysis:

Results of Statistical Analysis:

A very significant difference (p=0.0037) between treatment and controlgroups in the progression of arthritis (t-test difference between meanseverity score from day one since incidence until day 19 sinceincidence).

Arthritis Incidence—In the control group, autoimmune arthritis developeda rate and incidence considerable normal for this strain of mouse(DBA/1). The control group achieved a 100% incidence by day 44. Theincidence of arthritis in the experimental group (peptide treated) wassomewhat lower than the control group, and the rate of arthritisdevelopment appeared to be delayed.

Likewise, the number of paws per mouse in the treatment group wassignificantly lower than control at a 95% confidence limit.

Severity of Arthritis—The severity of arthritis was analyzed on thebasis of degree of inflammation (scored as described above) and thenumber of affected limbs. As seen in the figure below, differencesbetween the two groups were clearly observed when analyzed as meanSeverity Score/Mouse. While these data are weighted somewhat by thedifferences in arthritis incidence, the differences in the severityappear to be even greater than the differences in incidence.

Confidence limits were drawn using a 95% t-statistic on the residualdistributions obtained by subtracting Actual-Expected readings. Expectedreadings were obtained using a linear regression model of the true data.

Weight loss is normally found in CIA and can therefore be a factor indetermining severity of disease. The weight gain of the control groupwas less than that of the treatment group. Therefore weight measurementwere compared between days 21 and day 46 and a Mann-Witney significancetest showed that the weight difference between the two samples (controlvs. treatment) marked as weight on day 46 divided by weight on day 21,is marginally significant (P<0.05, two-tailed test). Mean weight gain(day 21 to 46) in control was 2.1% and in treatment group was 5.4%.

Results of the statistical analysis for arthritis paw severity andincidence are shown in FIGS. 1 to 6.

Conclusion and Discussion:

The above-described results in FIGS. 1-6 clearly demonstrate for thefirst time relative to the prior art, treatment of a disease using AMPsand in particular, cathelicidin. Specifically, the above describedresults clearly demonstrate for the first time relative to the prior artoptimal in-vivo treatment in a mouse model for arthritis, which isassociated with inflammation and an autoimmune disease.

Cathelicidin significantly lowers incidence rate as well as severity ofarthritis in model for Collagen induced arthritis (p=0.025).

This experiment shows that intravenous or subcutaneous or IP injectionof cathelicidin is a viable mode of treatment for arthritis, rheumaticdiseases and connective tissue/inflammatory diseases include arthritis,rheumatoid arthritis, pyogenic arthritis, mixed connective tissuedisease, cholesteatoma, relapsing polychondritis, autoimmune myositis,primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis,tendinitis, a ligament inflammation, chondritis, a joint inflammation, asynovial inflammation, carpal tunnel syndrome, osteoarthritis,ankylosing spondylitis, a skeletal inflammation, an autoimmune eardisease, osteoporosis, fibromyalgia, periodontitis, and an autoimmunedisease of the inner ear.

This experiment also shows that oral, intravenous or subcutaneous or IPinjection of cathelicidin forms a viable mode of treatment for therelated inflammatory systemic diseases include systemic lupuserythematosus, systemic sclerosis, septic shock, toxic shock syndrome,Reiter's syndrome, and cachexia.

Example 2 Cathelicidin for the Treatment of Multiple Sclerosis and CNSInflammatory Disease

Multiple sclerosis (MS) is an immune-mediated demyelinating disease ofthe central nervous system (CNS) of unknown etiology.

Cathelicidin is expressed in the CNS. In this experiment delivery of thecathelicidin was made by injection (IP).

Materials and Methods:

Protocol for Myelin Oligodendrocyte Protein (MOG)-peptide induced EAE inC57BL/6 mice.

Mice.

C57BL/6 (B6) mice were purchased from Harlan (Jerusalem, Israel).Female, 9 week old mice were used in the experiment. The mice werehoused in the specific-pathogen free (SPF) animal facility of the HebrewUniversity and all experiments were approved by the institutional animalcare and use committee (IACUC).

Induction of EAE

Emulsion preparation: MOGB35-55B peptide (MEVGWYRSPFSRVVHLYRNGK) 1.25mg/ml in PBS was emulsified in complete Freund's adjuvant (CFA)supplemented with 400 ug M. tuberculosis (Mt) H37RA (Difco). Mice wereimmunized s.c. in the flank with 250 ug MOGB35-55B/CFA using a 25Gneedle.

200 ng Pertussis Toxin (Sigma) was injected i.v. at the time ofimmunization and 48 h later.

EAE Score

EAE was scored on a scale of 0-6: 0, no impairment; 1, limp tail; 2,limp tail and hind limb paresis; 3, ≧1 hind limb paralysis; 4, full hindlimb and hind body paralysis; 5, hind body paralysis and front limbparesis; 6, death.

EAE Treatment

Mice were treated with cathelicidin peptide of sequenceGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ (SEQ NO 61) was purchased andsupplied by Biosight Ltd of Karmiel, Israel and diluted in PBS, vs. PBSas a control. The cathelicidin was diluted in sterile PBS and divided toaliquots kept at −20° C. such that each aliquot was thawed once for use.Mice were treated by intraperitoneal (i.p.) injection of roughly 200 ulvolume (adjusted for weight) 3 times a week (Sun-Tues-Thurs) startingthe day of immunization with MOG/CFA and through day 48. Clinical EAEscores were evaluated through day 60.

Results:

Results are displayed in FIGS. 7, 8, 9,

A graph showing clinical score up to day 50 is shown as graph in FIG. 8.

Conclusion:

Cathelicidin peptide treatment lowered EAE severity and protected micefrom fatal EAE observed at a late stage of the disease in controlanimals. The lower dose of peptide, 0.2 mg/Kg was more protective thanthe higher 2 mg/Kg dose.

Cathelicidin or its analogs or fragments can therefore be used as a drugfor the treatment of neurological and CNS inflammatory diseases.

These include neurological/inflammatory diseases includeneurodegenerative disease, multiple sclerosis, Alzheimer's disease,Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barresyndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome,paraneoplastic neurological disease, paraneoplastic cerebellar atrophy,non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy,Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea,Gilles de la Tourette syndrome, autoimmune polyendocrinopathy, dysimmuneneuropathy, acquired neuromyotonia, arthrogryposis multiplex, opticneuritis, spongiform encephalopathy, migraine, headache, clusterheadache, and stiff-man syndrome.

Example 3 Development of a Fully Humanized Antibody to LL-37

A fully humanized antibody Single Chain Variable Fragment (scFv) to thecathelicidin LL-37 was developed using the two-hybrid system in yeast, atechnology as described in U.S. Pat. No. 6,610,472.

Briefly, a library of expression vectors was generated in yeast cellsthrough homologous recombination; and the encoded proteins complexeswith high binding affinity to their target molecule LL-37 was selectedby high throughput screening in vivo or in vitro. Testing for ability toinhibit LL-37 in-vivo was performed by measuring the ability of thehumanized antibody to inhibit bacterial killing by LL-37.

FIG. 10 shows a Western blot analysis of 4 different scFv developed thatbind LL-37.

FIG. 11 shows the inhibitory effect of scFv on LL-37 in a bacterialkilling assays. In order to find out the concentration of LL37 at which50% of the bacteria could be killed (called “IC50”). Basically theactivity protocol follows the ability of the antibody to block theantimicrobial activity of LL-37. The bacteria used was Pseudomonas thatwas isolated from a wound. The growth medium was LB. LL-37 was added ata concentration of 100 microgram/ml (the final volume or the reaction is50 microliter). Blocking antibodies at 1 or 5 microliter of antibody(=1:50 or 1:10 dilutions respectively. Low antibody levels ensure anon-specific effect. A 2nd fraction from the elution with 100 mMimidazole was used.

The antibody and LL-37 mixture was incubated at room temperature for 30minutes.

The bacteria were added (volume of 40 microliters). The mixture wasincubated shaking for 3 hours at 37 degrees. At that point LB was addedto maintain the growth since the volumes we used were so small in orderto grow the bacteria for longer incubation times, the mixture wasfurther incubated for additional 2-3 hours. Concentration of bacteriawas estimated by optical density (OD) reading at 490.

Example 4 Cathelicidin in the Treatment, Diabetes and Related DiseasesIncluding

Hyperglycemia or Hypoglycemia, hypotension, hypertension,glandular/inflammatory diseases obesity, atherosclerosis and diabetesrelated diseases such as periodontitis and diabetes associated healingdeficiencies or wounds.

Background:

TLR4 and CD14 are the receptor for LPS and play a critical role ininnate immunity. Stimulation of TLR4 activates pro-inflammatory pathwaysand induces cytokine expression in a variety of cell types. Inflammatorypathways are activated in tissues of obese animals and humans and playan important role in obesity-associated insulin resistance. TLR4 andCD14 are a molecular link among nutrition, lipids, and inflammation andthat the innate immune system participates in the regulation of energybalance and insulin resistance in response to changes in the nutritionalenvironment. (Hang Shi et al. The Journal of Clinical InvestigationVolume 116 Number 11 Nov. 2006) In a paper published (Diabetes56:1761-1772, 2007), It was shown that metabolic Endotoxemia InitiatesObesity and Insulin and it was suggested that lowering plasma LPSconcentration could be a potent strategy for the control of metabolicdiseases including insulin resistance. Therefore, the present experimentshows that insulin resistance and thereby glucose levels can becontrolled using cathelicidin and is therefore a novel drug for thetreatment of diabetes and diabetes related diseases. The in-vivo mousemodel used is as described in Biochemical and Biophysical ResearchCommunications 361 (2007) 140-145, “LPS-induced biomarkers in mice: Apotential model for identifying insulin sensitizers”. Lipopolysaccharide(LPS)-mediated inflammatory response may modulate pathways implicated ininsulin resistance (J Clin Endocrinol Metab 85: 3770-3778, 2000).

Materials and Methods:

Two groups of 6 mice were used. One group was treated with PBS and theother group with the mCRAMP cathelicidin peptide at 0.4 mg/Kg, bothgroups injected (IP) three times a week on Sunday Tuesday and Thursday.Both groups were fed on a high fat diet of 60% Kcal fat diet (ResearchDiets Inc. New Brunswick USA) for a four week period by which time undernormal circumstances they would be insulin resistant. At the end of fourweeks blood glucose was determined using a glucometer on blood drawn bytail-nicking of mice.

LPS was administered to C57BL/6 mice at 0.2 mg/kg. Mice were bledapproximately 2 h after LPS injection (T=0). Changes in insulindependent (or non-insulin dependent) sensitivity in regulating theglucose uptake were examined by calculating the linear slope of the fallor gain in glucose. Such a slope/gradient shows the rate of decrease ofglucose over time.

mCRAMP sequence: (SEQ NO 61) GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ.

In calculating the statistics, for each individual mouse, the glucoselevel at time T=2 hrs. was divided by the glucose level at time T=0 toobtain a ratio at T=2 hrs for each mouse. An average was calculated forall the ratios and a students t-test was performed.

Results:

A significant difference rate of change in glucose levels was noted(students t-test<0.05). Two hours following LPS administration, averageglucose levels in the control mice rose by 5.31% whereas average glucoselevels in the cathelicidin treated mice came down to 90.05% of theirinitial level of 2 hours previously.

The treatment group was protected from insulin insensitivity therebyleading to a reduction of glucose levels during the two hour period ascompared to the control group. The control group being insulin resistantdue to the high fat diet remained at high glucose level. Cathelicidinprotected the treatment group mice from insulin resistance normallydeveloped as by the control group over the four week period of high fatdiet. A graphic representation of the data is shown in FIG. 12. Agraphic representation of the data is shown in FIG. 12

Conclusion:

This experiment shows that intravenous or subcutaneous or IP injectionof Cathelicidin, its analogs or fragments inhibits insulin resistanceand hyperglacemia, as well as LPS induced disregulation of glucoselevels in blood, and can therefore be used for the treatment of diseasessuch as metabolic diseases or a glandular/inflammatory diseasesincluding: type I diabetes, type II diabetes, type B insulin resistance,Schmidt's syndrome, Cushing's syndrome, thyrotoxicosis, benign prostatichyperplasia, pancreatic disease, Hashimoto's thyroiditis, idiopathicadrenal atrophy, Graves' disease, androgenic alopecia, thyroid disease,thyroiditis, spontaneous autoimmune thyroiditis, idiopathic myxedema,ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmuneprostatitis, Addison's disease, and Type I autoimmune polyglandularsyndrome Diabetes mellitus and Type II diabetes, obesity, Hyperglycemiaor Hypoglycemia, complications of diabetes including skin ulcerations,and diabetes related eye diseases such as Proliferative retinopathy

Example 5 Cathelicidin in the Treatment of Obesity and Overweight asWell as Related Diseases Such as Periodontitis and Diabetes AssociatedDiseases and Healing Deficiencies

Background:

A high-fat diet chronically increased insulin resistance, obesity andmetabolic diseases. Diabetes and obesity are two metabolic diseasescharacterized by insulin resistance and low-grade inflammation.

The present experiment shows that obesity can be controlled usingcathelicidin or cathelicidin fragments or analogues and is therefore anovel drug for the treatment of obesity and obesity related diseases.The in-vivo mouse model used is as described in (Diabetes 56:1761-1772,2007).

Materials and Methods:

Two experiments were performed, one on a regular diet and one on ahigh-fat diet:

In the first experiment:

Briefly, mice were fed on a normal non-high-fat diet for 21 days andtheir average weight was monitored. Two groups of Male DBA/1 mice, 10mice in each group (treatment & Control) with an average age of 10 weeksin each group.

Control group were injected with vehicle (150 mM saline) whilst theexperimental group were injected with the cathelicidin mCRAMP at aconcentration of 1.5 mg/kg on day 0. Subsequently on days 2 and 4, thedose was reduced to 1.0 mg/kg. Starting with day 7 and through to day21, a dose of 0.8 mg/kg was used. All treatments were performed 3 timesper week, on a Monday, Wednesday, and Friday schedule, and the peptideor control vehicle was administered intraperitoneally for eachtreatment, rotating injection areas. Results shown in FIG. 13.

mCRAMP sequence is: (SEQ NO 61) GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ

In the second experiment:

Briefly, Experimental obesity was induced in C57BL/6 mice by maintainingthem on a 60% Kcal fat diet (Research Diets Inc. New Brunswick USA) fora six week period.

The experiment contained 2 groups of 6 mice: Group 1: PBS, Group 2:mCRAMP cathelicidin 0.2 mg/Kg for 3 weeks and then 0.4 mg/Kg for another3 weeks. Mice were treated by intraperitoneal injection of PBS vs.peptide (200 1 per injection) on Sunday, Tuesday, and Thursday of eachweek. Mice were weighed at baseline and three times a week on each dayof treatment.

Results:

In the first experiment using a non-high-fat diet, and as shown in FIG.13, mice in treatment group increased in weight at a rate of 0.0536gm/day while in the treatment the weight gain was 0.0488 gm/day.

In the second experiment using a high-fat diet the average weight in thecontrol was divided by the average weight in the treatment groups foreach of the readings (three per week). A trend was seen in the graphplotted as seen in FIG. 14. This trend is significant when analyzingusing a statistical test of linear regression and residual analysis.

Continuing the treatment to day 50, a statistically significantdifference between the two groups was noted by using a students t-test(<0.05) after comparing the weights of the two groups on day 50.

Therefore, treatment over long term using cathelicidin at a normallyendogenous level would significantly reduce weight in obese mice.

Conclusion:

This experiment shows that intravenous or subcutaneous or IP injectionof cathelicidin is a viable mode of treatment for obesity. At a 60% Kcaldiet, a dosage of 0.4 mg/Kg three times a week was enough tosignificantly reduce weight in obese mice and prevent obesity.

Conclusion:

This experiment shows that intravenous or subcutaneous or IP injectionof cathelicidin is a viable mode of treatment for obesity and that itseffect is dose dependant. At a 60% Kcal diet, a dosage of 0.4 mg/Kgthree times a week was enough to significantly reduce weight in obesemice and prevent obesity.

Example 6

Use of Cathelicidin for treating osteoporosis, ankylosing spondylitis,osteoarthritis and periodontitis by preventing bone erosion orresorption.

Background:

Vitamin D3, a commonly used medication for osteoporosis also induces theexpression of cathelicidin through the calcitriol/VDRE. For this reasoncathelicidin was studied on its effect on bone resorption, degradationor formation.

Bone erosion or degradation in rheumatoid arthritis, periodontitis andosteoarthritis is a result of persistent chronic inflammation. Likewise,in osteoporosis there exists an imbalance between bone resorption andbone formation. This imbalance is due to process by which osteoclastcell activity, the process that breaks down bone, dominates osteoblastcell activity, the process by which bone formation is performed.

Therefore the present experiment tested to see if there was anydifference in the bone degradation, inflammation or resorption statusbetween arthritic paws of control versus cathelicidin treated mice inthe mouse model of collagen induced arthritis as well as between thetreatment and control groups of LPS induced bone loss in periodontitis(J Clin Periodontol 2004; 31: 596-603). Also tested and observed werenon-inflamed joints and bone of control versus non-inflamed treatmentjoints and bone.

Cathelicidin was injected (IP) into treatment mice and compared withcontrol. Histological samples of bone taken from the ankle joints ofmice paws were analyzed and osteoclasts were counted using the H&E(hematoxylin and eosin stain) Immunohistochemical staining and with TRAPstaining technique (Acid Phosphatase, Leukocyte—Procedure No, 387 A fromSigma-Aldrich).

Eight groups were analyzed according to their inflammatory status:

Two groups: control and treatment groups had induced inflammation intheir paws but their inflammation levels were similar.

Two further groups: control and treatment groups had no inflammation intheir paws.

Two further groups: control and treatment groups had inducedinflammation in their paws but their inflammation levels weredissimilar.

Two further groups: control and treatment groups having induced lowgrade inflammation via LPS injections (IP) were studied for bonemorphology differences in mandibles.

By this method, it was possible to observe bone degradation,inflammation or resorption by monitoring osteoclast and immune orinflammatory cell activity.

Materials and Methods:

Paws from the Collagen induced arthritis (CIA) experiment were studied.In all 80 paws from 20 mice were available for study and of those, only15 were studied according to their inflammation/arthritic severityscore. The protocol for induction of the CIA is reported in experiment 1above.

Histology:

For the detection of TRAP+ cells in histological slides of joints,amputated limbs were fixed in 1% paraformaldehyde for several weeks andwashed with PBS. The tissues were decalcified by incubation in 0.5 MEDTA/PBS, pH 7.4, for 10 days, in which the EDTA solution was changedevery day. Tissues were embedded in paraffin and 6 μm sections weremade. Deparaffinised, rehydrated sections were either stained withhaematoxylin and eosin or preincubated for 2.5 hours at 37° C. in a 12.5mM sodium tartrate solution in 100 mM acetate buffer, pH 5.5.

Subsequently, sections were incubated for 1 hour at 37° C. in acidphosphatase substrate solution (0.05% naphthol AS-BI phosphate 50 mMsodium tartrate, 0.16% p-rosanilin, 0.16% NaNO2, 25% Michaelis' 0.14 Macetate/barbital buffer, pH 5.0, in distilled water). Sections werewashed with distilled water, counterstained with 0.15% Lightgreen SFYellowish in 0.2% acetic acid, incubated for 10 s in 1% acetic acid anddried at 37° C. Red-staining cells were considered to contain TRAP, andTRAP+ multinucleated cells (three or more nuclei) were regarded asosteoclasts.

Paws having similar arthritic scores for equal lengths of time werecompared for bone resorption and degradation in cathelicidin treatmentgroup versus control group. This type of comparison rules out anyinfluence of inflammation as a determinant of bone degradation orankylosis leaving the differentiation status of osteoblasts andosteoclasts as the main influence.

The materials and methods used for inducing arthritic bone degredationare described for the mouse model in example 1 above. The arthritic pawsof grade 3 severity index and above were obtained from this sameexperiment and placed in fixative for histology measurements.

Experimental Results:

For observation of Osteoclasts and Inflammatory bone degradationincluding periodontitis, arthritis osteoarthritis, slides are stainedwith H&E and with TRAP. Several paws having similar severity index atequal duration were histologically examined (see table of FIG. 15. Inaddition, non-inflammed paws in cotrol and treatment groups were alsostudies (FIG. 15). Other paws having different severity index anddurations were also studied. In the figures, LF=Left Front paw, RF=RightFront paw, LH=Left Hind paw, and RH=Right Hind paw.

A clear difference in erosion and resorption between treatment andcontrol groups was noted with less degradation and less resorptionobserved in treatment group. Degradation or deformation was mainly seenin control group. Likewise for mice chosen as having no difference inseverity index and duration of arthritic paws, there was a similardistinct difference in bone resorption/erosion between the two groups.

FIG. 16 is an example of histology slides between mouse 3 (Right Front)and FIG. 17 shows the H&E staining of mouse 3 (Right Front) paws.

FIG. 18 shows the TRAP staining of control mouse 13 having noinflammatory sign in its paw yet still showing more osteoclasts that theinflamed mouse 3 shown above.

FIG. 19 shows a TRAP staining of inflamed paw of control mouse 17—Rightfront paw clearly showing a marked increase in osteoclasts.

Clearly, the control mouse has a higher number of active osteclasts aswell as higher resorption and degradation even though both mice have thesame inflammatory status.

Conclusion and Discussion:

Cathelicidin, inhibits bone erosion and deformation as found in eitherosteoporosis, ankylosing spondylitis, osteoarthritis and periodontitisand can therefore be used as a drug for treating these diseases.

In the present experiment, cathelicidin was delivered by IP injections.Therefore, it is obviously implied that the drug delivery ofcathelicidin can be either orally using a vehicle carrier to the bloodsteam via the GI tract or by injection i.v. or subcutaneous injections.

The data convincingly shows that Cathelicidin is a suitable drugcandidate in the treatment of osteoporosis, ankylosing spondylitis,osteoarthritis and periodontitis, Osteomyelitis, bone cancer,Osteogenesis imperfecta, Paget's disease, Osteochondroma, Osteomalacia,Osteomyelitis, Osteopetroses, Renal Osteodystrophy, Unicameral BoneSpurs, Bone Tumor, Craniosynostosis, Enchondroma, Fibrous Dysplasia,Giant Cell Tumor of Bone, Infectious Arthritis, Osteomyelitis,Klippel-Feil Syndrome, Limb Length Discrepancy, OsteochondritisDissecans, and bone loss in periodontitis.

Example 7 Use of Cathelicidin Analog and Fragment for Optimal Treatmentof Diseases, Such as Psoriasis, which are Associated with Inflammation,Autoimmunity and/or Skin Cell/Tissue Proliferation/DifferentiationImbalance and Wound Healing

Background:

Diseases associated with inflammation, autoimmunity and/or skincell/tissue proliferation/differentiation imbalance include numerousdiseases, such as psoriasis and dandruff, for which no optimal therapyexists. Angiogenesis and epithelialization common in psoriatic skin isenhanced by AMPs such as LL-37 (Koczulla, R. et al., 2003. J. Clin.Invest 111:1665-1672; Heilborn, J D. et al., 2003. J Invest Dermatol120:379-389). An optimal strategy for treating such diseases would be toidentify factors involved in dysregulation of skin cell/tissueproliferation/differentiation, and to use compounds capable ofinhibiting the activity of such factors to treat such diseases.

Human tissue kallikreins are a family of 15 trypsin-like orchymotrypsin-like secreted serine proteases (KLK1-KLK15). Multiple KLKshave been quantitatively identified in normal stratum corneum (SC) andsweat as candidate desquamation-related proteases. Aberrant human tissuekallikrein levels levels in the stratum corneum and serum of patientswith psoriasis (British Journal of Dermatology 2007 156, pp875-883).These kallikreins are protease involved in the maturation process ofcathelicidin LL-37 from its precursor hCAP-18. Inappropriate balancebetween various proteases can be a determining factor as to whethercathelicidin is cleaved into its pro-inflammatory or to itsanti-inflammatory fragments.

As was demonstrated by the present inventor in WO 2004-056307,cathelicidin is an immune regulator in-vivo and plays a major role inpsoriasis and skin inflammation. Inhibiting or regulating its activityis essential for treatment of the disease Inhibition of cathelicidin inskin inflammation was further demonstrated in psoriasis (Nature 2007Oct. 4; 449(7162):564-9) and in other skin inflammatory diseases such asrosacea (Nat Med. 2007 August; 13(8):975-80).

While reducing the present invention to practice, a method of usingdominant negative cathelicidin peptide or fragments for optimaltreatment in a human of a disease associated with inflammation,autoimmunity and/or skin cell/tissue proliferation/differentiationimbalance, such as psoriasis, was demonstrated for the first time, asdescribed below, thereby overcoming the limitations of the prior art.

Materials and Methods:

Antimicrobial peptides (AMPs): The antimicrobial peptides used were usedwere the fragment cathelicidin SK29:S KEKIGKEFKRIVQRIKDFLRNLVPRTES orGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ (SEQ NO 61) the mouse cathelicidinCRAMP (BIOSIGHT LTD, Karmiel, Israel),

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 14)_(—) Catalogue No.61302

LL-37 antimicrobial peptide human, AnaSpec, Inc. USA., HumanBeta-Defensin-2 peptide was purchased from Sigma-Aldrich.

Human in-vivo psoriatic lesion treatment: Cathelicidin 10 ug/ml or humanbeta defensin-2 diluted in PBS containing 0.1% BSA, or buffer carrierwas applied to lesions in a human subject in a blind trial.

Experimental Results:

Whereas the human beta defensin-2 showed a worsening of skin psoriaticlegion over a seven week course of treatment, as exemplified in FIG. 20,the cathelicidins LL-37 and SK29 showed a slight improvement over thecourse of 5 days treatment.

Conclusion and Discussion:

Recently published material regarding rosacea has shown (Nat Med. 2007August; 13(8):975-80) that inappropriate cathelicidin processing byendogenous protease is responsible for the disease progression ofrosacea. It may well be that similar mechanisms are in effect in otherdiseases such as psoriasis in which case dominant negative peptideinhibitors that compete with fragments of LL-37 whithout activating thedisease would form viable modes of treatment for the disease.

There is no contradiction that both inhibiting LL-37 by an antibody aswas demonstrated by the previous application of the inventors in WO2004-056307 and in the publication (Nature 2007 Oct. 4; 449(7162):564-9)and making use of LL-37 can be similarly effective. One possibility isthat LL-37 may inhibit its own fragments formed by inappropriateendogenous protease action.

Example 8 Use of Cathelicidin Fragments or Analogs for the Treatment ofDiabetes

In-Vitro Studies on Beta-Cells

Background:

No optimal therapy exists for treatment of type 1 diabetes. An optimalstrategy for treating such a disease would be to identify factorsinvolved in inducing beta cell growth. While reducing the presentinvention to practice, a significant role for AMPs in driving beta cellproliferation was identified, and the capacity of cathelicidin to inducegrowth so as to enable optimal treatment of type 1 diabetes wasdemonstrated, as described below, thereby overcoming the limitations ofthe prior art.

Materials and Methods:

Antimicrobial Peptides (AMPs):

The antimicrobial peptides human cathelicidin LL-37:LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 14) was obtained fromAnaSpec, USA (Catalogue numbers: 61302). Cathelicidin at 2 ug/ml wasadded to the plate and compared with control.

Thymidine Incorporation Cell Proliferation Assay:

Cell proliferation was evaluated by measuring [3(H)]-thymidineincorporation into DNA. Cells were pulsed with [3(H)]-thymidine (1microcurie/mL, ICN, Irvine, Calif.) for 1 hour, at 37 degreescentigrade. After incubation, cells were washed 3 times with PBS,incubated for 15 minutes at room temperature in 5% trichloroacetic acidand solubilized in 1% triton X-100. The radioactivity incorporated intothe cells was counted in the [3(H)]-window of a Tricarb liquidscintillation counter. Mean values were determined from measurements oftriplicate samples under each experimental condition for eachexperiment. Thymidine incorporation was determined as number ofdisintegrations per minute (DPM) per mg of protein.

Experimental Results:

Beta Cells are Significantly Stimulated to Proliferate by AMPs:

In order to investigate the effects of AMPs on beta cell growth, mouseBETATC beta cell line were chronically treated with cathelicidin, andtheir proliferation was monitored. As can be seen in FIG. 21, in allskin epithelial cells exposure to cathelicidin led to a slight increasein cell proliferation. This data clearly demonstrates that AMPs areinvolved in the pathogenesis of diabetes and in particular to type 1diabetes with respect to cellular hyperproliferation.

Conclusion:

The above-described results clearly demonstrate that AMPs, such ascathelicidin or its fragments or its analogs, are involved in drivingproliferation of beta cells and therefore can be used in the treatmentof diabetes by enabling the presence of an increased number of insulinsecreting beta cells.

Example 9 Chemotaxis Assays

Chemotaxis assays. Cells (e.g. neutrophils, monocytes, T cells, HEK293;25 microliters at a density of 1.0-3.0×106 cells/ml) in RPMI medium(Beit Haemek) containing 0.5% BSA (Sigma-Aldrich) are placed on the topof a 96-well ChemoTx disposable chemotaxis apparatus with a 5 micronpore size (Neuroprobe). Tenfold serial dilutions of the tested reagentin RPMI medium with or without 0.5% BSA are placed in the bottom wellsof the chamber. The apparatus is incubated for 60-600 min at 37° C. inan atmosphere of 5% carbon dioxide, and the cells migrating at eachconcentration of chemoattractant is counted with the use of an invertedmicroscope.

Cells (1×107/mL) are suspended in a buffer containing 0.25% BSA, 145 mMNaCl, 5 mM KCl, 10 mM Na/MOPS, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10mM HEPES (all from Sigma-Aldrich), pH 7.4, and incubated with 2micromolar Fura-2-AM (Molecular Probes, Eugene, Oreg.), for 40 min atroom temperature. The cells are washed once, resuspended in the buffercontaining 0.25% BSA, and are kept at room temperature. Just before use,aliquots of the cells (4×105) are washed and resuspended in 2 ml buffercontaining 0.05% BSA in a stirred cuvette at 37° C. Measurement ofintracellular Ca2+ concentration and chemotaxis assays are performed aspreviously described (Maghazachi, A A. et al., 1997. FASEB J.11:765-774)

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, and patentapplications and sequences identified by their accession numbersmentioned in this specification are herein incorporated by reference intheir entireties into the specification, to the same extent as if eachindividual publication, patent, or patent application or sequenceidentified by its accession number was specifically and individuallyindicated to be incorporated herein by reference. In addition, citationor identification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

1. A method for treating in a mammal a metabolic disease or disorder,selected from the group consisting of Type 2 diabetes, insulinresistance, hyperglycemia, metabolic syndrome, obesity, overweight,cardiovascular disease, dyslipidemia, and metabolic endotoxemia themethod comprising administering to a subject in need thereof, atherapeutically effective amount of a cathelicidin peptide selected frommCRAMP (SEQ ID NO: 61) or LL-37 (SEQ ID NO: 14).
 2. The method of claim1, wherein the mammal is a human.
 3. The method of claim 1, wherein thecathelicidin peptide is mCRAMP (SEQ ID NO:61).
 4. The method of claim 1,wherein the cathelicidin is LL-37 (SEQ ID NO: 14).
 5. The method ofclaim 1, wherein the method is a method of treating insulin resistance.6. The method of claim 1, wherein the method is a method of treatingObesity or overweight.
 7. The method of claim 1, wherein the method is amethod of treating diabetes.
 8. The method of claim 1, wherein themethod is a method of treating metabolic syndrome.